Search

We determined prognostic implications of acute lung injury (ALI) and organizing pneumonia (OP), including timing relative to transplantation, in a multicenter lung recipient cohort. We sought to understand clinical risks that contribute to development of ALI/OP. We analyzed prospective, histologic diagnoses of ALI and OP in 4786 lung biopsies from 803 adult lung recipients. Univariable Cox regression was used to evaluate the impact of early (≤90 days) or late (90 days) posttransplant ALI or OP on risk for chronic lung allograft dysfunction (CLAD) or death/retransplantation. These analyses demonstrated late ALI/OP conferred a two- to threefold increase in the hazards of CLAD or death/retransplantation; there was no association between early ALI/OP and these outcomes. To determine risk factors for late ALI/OP, we used univariable Cox models considering donor/recipient characteristics and posttransplant events as candidate risks. Grade 3 primary graft dysfunction, higher degree of donor/recipient human leukocyte antigen mismatch, bacterial or viral respiratory infection, and an early ALI/OP event were significantly associated with increased late ALI/OP risk. These data from a contemporary, multicenter cohort underscore the prognostic implications of ALI/OP on lung recipient outcomes, clarify the importance of the timing of these events, and identify clinical risks to target for ALI/OP prevention.

First-line therapy for patients with metastatic NSCLC includes checkpoint inhibitor monotherapy, dual checkpoint inhibition, or combination with chemotherapy. We compared outcomes with combination chemoimmunotherapy versus dual checkpoint inhibition as first-line treatment for patients with metastatic NSCLC.This open-label, randomized clinical trial was conducted at 44 sites in Canada and Australia. Patients with treatment-naive, metastatic NSCLC without sensitizing EGFR or ALK alterations were randomized (1:1) to receive treatment with durvalumab plus tremelimumab with or without platinum-doublet chemotherapy. The primary end point was overall survival (OS). Secondary end points were progression-free survival, overall response rate, and safety.A total of 301 patients were randomized. Median OS was 16.6 months (95% confidence interval [CI]: 12.6-19.1) with chemotherapy plus immunotherapy and 14.1 months (95% CI: 10.6-18.3) with immunotherapy (hazard ratio = 0.88, 90% CI: 0.67-1.16, p = 0.46). Median progression-free survival with chemotherapy plus immunotherapy was 7.7 months (95% CI: 5.5-8.5) and 3.2 months (95% CI: 2.7-5.1) with immunotherapy (hazard ratio = 0.67, 95% CI: 0.52-0.88). The overall response rate with chemoimmunotherapy was 42.4% and 29.3% with immunotherapy (adjusted OR = 1.69, 95% CI: 1.04-2.76). The percentage of patients with grade 3 or higher adverse events was 82% in the chemotherapy plus immunotherapy group and 70% in the immunotherapy group. Exploratory analyses of programmed death-ligand 1 expression and blood-based tumor mutation burden revealed no differential treatment effect on OS.The addition of chemotherapy to durvalumab plus tremelimumab in the first-line treatment of stage IV NSCLC did not improve survival compared with durvalumab plus tremelimumab alone. Further study is warranted to identify patients that benefit from initial immunotherapy alone versus combination chemotherapy plus immunotherapy as first-line treatment.

Purpose: Molecular testing is critical to guiding treatment decisions in vulnerable patients with metastatic non-small cell lung cancer (mNSCLC), with testing delays associated with prolonged treatment times and higher mortality. Here, we evaluated the time from initial mNSCLC diagnosis to treatment decision (TTD) following implementation of in-house EGFR, ALK, and PD-L1 testing at our institution. Methods: We conducted a retrospective chart review of 165 patients (send-out testing, n=92; in-house testing, n=73) with newly diagnosed stage IV NSCLC treated at our institution. Data were compared during the send-out (March 2017-May 2019) and in-house (July 2019-March 2021) testing periods. We performed a detailed workflow analysis to provide insight on the pre-analytic, analytic, and post-analytic intervals that constituted the total TTD. Results: TTD was significantly shorter with in-house testing (10 days vs.18 days, pvs. 3 days, pvs. 8 days, pvs.74% send-out, pvs. 52% send-out, pvs. 86% send-out, p=0.57). Conclusion: Our results demonstrate the advantages of in-house biomarker testing for mNSCLC at high-volume oncology centres. Incorporation of in-house testing may reduce barriers to offering personalized medicine by improving the time to optimal systemic therapy decision.

Background It is estimated that millions of North Americans would qualify for lung cancer screening and that billions of dollars of national health expenditures would be required to support population-based computed tomography lung cancer screening programs. The decision to implement such programs should be informed by data on resource utilization and costs. Methods Resource utilization data were collected prospectively from 2059 participants in the Pan-Canadian Early Detection of Lung Cancer Study using low-dose computed tomography (LDCT). Participants who had 2% or greater lung cancer risk over 3 years using a risk prediction tool were recruited from seven major cities across Canada. A cost analysis was conducted from the Canadian public payer's perspective for resources that were used for the screening and treatment of lung cancer in the initial years of the study. Results The average per-person cost for screening individuals with LDCT was USD453 (95% confidence interval [CI], USD400–USD505) for the initial 18-months of screening following a baseline scan. The screening costs were highly dependent on the detected lung nodule size, presence of cancer, screening intervention, and the screening center. The mean per-person cost of treating lung cancer with curative surgery was USD33,344 (95% CI, USD31,553–USD34,935) over 2 years. This was lower than the cost of treating advanced-stage lung cancer with chemotherapy, radiotherapy, or supportive care alone, (USD47,792; 95% CI, USD43,254–USD52,200; p = 0.061). Conclusion In the Pan-Canadian study, the average cost to screen individuals with a high risk for developing lung cancer using LDCT and the average initial cost of curative intent treatment were lower than the average per-person cost of treating advanced stage lung cancer which infrequently results in a cure.

ChronicPseudomonas aeruginosa(Pa) lung infections are the leading cause of mortality among cystic fibrosis (CF) patients; therefore, the eradication of new-onset Pa lung infections is an important therapeutic goal that can have long-term health benefits. The use of early antibiotic eradication therapy (AET) has been shown to eradicate the majority of new-onset Pa infections, and it is hoped that identifying the underlying basis for AET failure will further improve treatment outcomes. Here we generated random forest machine learning models to predict AET outcomes based on pathogen genomic data. We used a nested cross validation design, population structure control, and recursive feature selection to improve model performance and showed that incorporating population structure control was crucial for improving model interpretation and generalizability. Our best model, controlling for population structure and using only 30 recursively selected features, had an area under the curve of 0.87 for a holdout test dataset. The top-ranked features were generally associated with motility, adhesion, and biofilm formation.AUTHOR SUMMARYCystic fibrosis (CF) patients are susceptible to lung infections by the opportunistic bacterial pathogenPseudomonas aeruginosa(Pa) leading to increased morbidity and earlier mortality. Consequently, doctors use antibiotic eradication therapy (AET) to clear these new-onset Pa infections, which is successful in 60%-90% of cases. The hope is that by identifying the factors that lead to AET failure, we will improve treatment outcomes and improve the lives of CF patients. In this study, we attempted to predict AET success or failure based on the genomic sequences of the infecting Pa strains. We used machine learning models to determine the role of Pa genetics and to identify genes associated with AET failure. We found that our best model could predict treatment outcome with an accuracy of 0.87, and that genes associated with chronic infection (e.g., bacterial motility, biofilm formation, antimicrobial resistance) were also associated with AET failure.

Antimicrobial susceptibility testing (AST) is essential for detecting resistance in Pseudomonas aeruginosa and other bacterial pathogens. Here we evaluated the performance of broth microdilution (BMD) panels created using a semi-automated liquid handler, the D300e Digital Dispenser (Tecan Group Ltd., CH) that relies on inkjet printing technology. Microtitre panels (96-well) containing nine twofold dilutions of 12 antimicrobials from five classes (β-lactams, β-lactam/β-lactamase inhibitors, aminoglycosides, fluoroquinolones, polymyxins) were prepared in parallel using the D300e Digital Dispenser and standard methods described by CLSI/ISO. To assess performance, panels were challenged with three well characterized quality control organisms and 100 clinical P. aeruginosa isolates. Traditional agreement and error measures were used for evaluation. Essential (EA) and categorical (CA) agreements were 92.7% and 98.0% respectively for P. aeruginosa isolates with evaluable on-scale results. The majority of minor errors that fell outside acceptable EA parameters (≥ ± 1 dilution, 1.9%) were seen with aztreonam (5%) and ceftazidime (4%), however all antimicrobials displayed acceptable performance in this situation. Differences in MIC were often log2 dilution lower for D300e dispensed panels. Major and very major errors were noted for aztreonam (2.6%) and cefepime (1.7%) respectively. The variable performance of D300e panels suggests that further testing is required to confirm their diagnostic utility for P. aeruginosa.

Immune checkpoint inhibitor (ICI) therapies have revolutionized the management of patients with NSCLC and have led to unprecedented improvements in response rates and survival in a subset of patients with this fatal disease. However, the available therapies work only for a minority of patients, are associated with substantial societal cost, and may lead to considerable immune-related adverse events. Therefore, patient selection must be optimized through the use of relevant biomarkers. Programmed death-ligand 1 protein expression by immunohistochemistry is widely used today for the selection of programmed cell death protein 1 inhibitor therapy in patients with NSCLC; however, this approach lacks robust sensitivity and specificity for predicting response. Tumor mutation burden (TMB), or the number of somatic mutations derived from next-generation sequencing techniques, has been widely explored as an alternative or complementary biomarker for response to ICIs. In theory, a higher TMB increases the probability of tumor neoantigen production and therefore, the likelihood of immune recognition and tumor cell killing. Although TMB alone is a simplistic surrogate of this complex interplay, it is a quantitative variable that can be relatively readily measured using currently available sequencing techniques. A large number of clinical trials and retrospective analyses, employing both tumor and blood-based sequencing tools, have evaluated the performance of TMB as a predictive biomarker, and in many cases reveal a correlation between high TMB and ICI response rates and progression-free survival. Many challenges remain before the implementation of TMB as a biomarker in clinical practice. These include the following: (1) identification of therapies whose response is best informed by TMB status; (2) robust definition of a predictive TMB cut point; (3) acceptable sequencing panel size and design; and (4) the need for robust technical and informatic rigor to generate precise and accurate TMB measurements across different laboratories. Finally, effective prediction of response to ICI therapy will likely require integration of TMB with a host of other potential biomarkers, including tumor genomic driver alterations, tumor-immune milieu, and other features of the host immune system. This perspective piece will review the current clinical evidence for TMB as a biomarker and address the technical sequencing considerations and ongoing challenges in the use of TMB in routine practice.

Background There is still controversy whether full lung palpation is required for patients undergoing pulmonary metastasectomy. We aimed to compare pulmonary ipsilateral recurrence (IR) after video-assisted thoracic surgery (VATS) or open surgery. Methods A retrospective study of all patients who underwent surgery for colorectal cancer lung metastases between 2003 and 2012 was performed. IR rate was compared between the 2 groups after adjusting for a propensity score matching based on age, sex, disease-free interval, number of metastases, type of resection, presence of a cardiovascular risk factor, presence of a respiratory risk factor, as well as the interaction between the number of metastases and the disease-free interval. The propensity score was used for matched and weighted comparisons of VATS and open patients. Results A total of 211 patients underwent surgery for colorectal cancer lung metastases. Of these, 75 (35.5%) were performed via VATS and 136 (64.5%) via open surgery. Before matching, 118 (55.9%) were male and the median age at the time of metastases diagnosis was 61 (range, 49.8-72.2) years. Median disease free-interval was 20 (19.7 ± 28.3) months; 22 (21.6 ± 28.5) months in VATS and 19 (19.0 ± 28.3) months in open surgery. In total, 19 (25.3%) developed IR in VATS, and 39 (28.7%) in open surgery. Five-year overall survival was 53.1% (61.9% VATS; 49.2% open). In the matched sample, IR was 23.6% in VATS vs 26.2% in open surgery (95% confidence interval for risk reduction with VATS: –22.6% to 17.5%; P = .80). Conclusions No significant difference was observed in IR rates between VATS and open surgery in the treatment of colorectal cancer lung metastases.

The recent development of immune checkpoint inhibitors (ICIs) has led to promising advances in the treatment of patients with NSCLC and SCLC with advanced or metastatic disease. Most ICIs target programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) axis with the aim of restoring antitumor immunity. Multiple clinical trials for ICIs have evaluated a predictive value of PD-L1 protein expression in tumor cells and tumor-infiltrating immune cells (ICs) by immunohistochemistry (IHC), for which different assays with specific IHC platforms were applied. Of those, some PD-L1 IHC assays have been validated for the prescription of the corresponding agent for first- or second-line treatment. However, not all laboratories are equipped with the dedicated platforms, and many laboratories have set up in-house or laboratory-developed tests that are more affordable than the generally expensive clinical trial–validated assays. Although PD-L1 IHC test is now deployed in most pathology laboratories, its appropriate implementation and interpretation are critical as a predictive biomarker and can be challenging owing to the multiple antibody clones and platforms or assays available and given the typically small size of samples provided. Because many articles have been published since the issue of the IASLC Atlas of PD-L1 Immunohistochemistry Testing in Lung Cancer, this review by the IASLC Pathology Committee provides updates on the indications of ICIs for lung cancer in 2019 and discusses important considerations on preanalytical, analytical, and postanalytical aspects of PD-L1 IHC testing, including specimen type, validation of assays, external quality assurance, and training.

Tissue contaminants in anatomical pathology are not uncommon. While issues related to the presence of extraneous tissue on glass slides are often easily resolved, this is not always the case and several factors may contribute to diagnostic difficulty. Because of this, familiarity with the different steps involved in handling specimens in the anatomical pathology laboratory is essential when troubleshooting possible cross-contaminants. Most commonly, the specimen constituting the source of cross-contamination is handled before the actual contaminated case; however, this is not always so. In this article, we review the steps involved in processing pathology specimens as they pertain to cross-contamination; share an approach covering how to troubleshoot and prevent tissue contaminants in a systematic and practical manner; present some examples from our own experiences; and compare our experience to what is reported in the literature. The information included in this article will be of use to all members of the anatomical pathology team including medical laboratory technologists, laboratory managers and supervisors, pathologist assistants, trainees in pathology, and pathologists.

Localized malignant mesotheliomas (LMM) is an uncommon and poorly recognized neoplasm. Its pathologic diagnosis is often surprising in patients with serosal/subserosal based localized tumors that are clinically suspicious for metastatic lesions or primary sarcomas. Once a tumor is diagnosed as "mesothelioma", LMM is often mistaken for diffuse malignant mesothelioma (DMM). Best currently available evidence about LMM was collected from the literature and cases diagnosed by members of the International Mesothelioma Panel (IMP). One hundred and one (101) LMM have been reported in the English literature. Patients had localized tumors with identical histopathologic features to DMM. Patients ranged in age from 6 to 82 years; 75% were men. Most (82%) of the tumors were intrathoracic. Others presented as intrahepatic, mesenteric, gastric, pancreatic, umbilical, splenic, and abdominal wall lesions. Tumors varied in size from 0.6 to 15 cm. Most patients underwent surgical resection and/or chemotherapy or radiation therapy. Median survival in a subset of patients was 29 months. Seventy two additional LMM from IMP institutions ranged in age from 28 to 95 years; 58.3% were men. Sixty tumors (83.3%) were intrathoracic, others presented in intraabdominal sites. Tumors varied in size from 1.2 to 19 cm. Median survival for 51 cases was 134 months. Best evidence was used to formulate guidelines for the diagnosis of LMM. It is important to distinguish LMM from DMM as their treatment and prognosis is different. A multidisciplinary approach is needed for the diagnosis of LMM as it shows identical histopathology and immunophenotype to DMM.

BackgroundIt 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.ResultsUsing 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.ConclusionRelative 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.

Introduction Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is required to determine the eligibility for pembrolizumab monotherapy in advanced NSCLC worldwide and for several other indications depending on the country. Four assays have been approved/ Communaute Europeene–In vitro Diagnostic (CV-IVD)–marked, but PD-L1 IHC seems diversely implemented across regions and laboratories with the application of laboratory-developed tests (LDTs). Method To assess the practice of PD-L1 IHC and identify issues and disparities, the International Association for the Study of Lung Cancer Pathology Committee conducted a global survey for pathologists from January to May 2019, comprising multiple questions on preanalytical, analytical, and postanalytical conditions. Result A total of 344 pathologists from 64 countries participated with 41% from Europe, 24% from North America, and 18% from Asia. Besides biopsies and resections, cellblocks were used by 75% of the participants and smears by 11%. The clone 22C3 was most often used (69%) followed by SP263 (51%). They were applied as an LDT by 40% and 30% of the users, respectively, and 76% of the participants developed at least one LDT. Half of the participants reported a turnaround time of less than or equal to 2 days, whereas 13% reported that of greater than or equal to 5 days. In addition, quality assurance (QA), formal training for scoring, and standardized reporting were not implemented by 18%, 16%, and 14% of the participants, respectively. Conclusions Heterogeneity in PD-L1 testing is marked across regions and laboratories in terms of antibody clones, IHC assays, samples, turnaround times, and QA measures. The lack of QA, formal training, and standardized reporting stated by a considerable minority identifies a need for additional QA measures and training opportunities.

Antimicrobial therapies against cystic fibrosis (CF) lung infections are largely aimed at the traditional, well-studied CF pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex, despite the fact that the CF lung harbors a complex and dynamic polymicrobial community. A clinical focus on the dominant pathogens ignores potentially important community-level interactions in disease pathology, perhaps explaining why these treatments are often less effective than predicted based on in vitro testing., Antimicrobial therapies against cystic fibrosis (CF) lung infections are largely aimed at the traditional, well-studied CF pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex, despite the fact that the CF lung harbors a complex and dynamic polymicrobial community. A clinical focus on the dominant pathogens ignores potentially important community-level interactions in disease pathology, perhaps explaining why these treatments are often less effective than predicted based on in vitro testing. A better understanding of the ecological dynamics of this ecosystem may enable clinicians to harness these interactions and thereby improve treatment outcomes. Like all ecosystems, the CF lung microbial community develops through a series of stages, each of which may present with distinct microbial communities that generate unique host-microbe and microbe-microbe interactions, metabolic profiles, and clinical phenotypes. While insightful models have been developed to explain some of these stages and interactions, there is no unifying model to describe how these infections develop and persist. Here, we review current perspectives on the ecology of the CF airway and present the CF Ecological Succession (CFES) model that aims to capture the spatial and temporal complexity of CF lung infection, address current challenges in disease management, and inform the development of ecologically driven therapeutic strategies.

Programmed death-ligand 1 (PD-L1) is used as a biomarker for anti-programmed cell death protein-1 (PD-1) or anti-PD-L1 immunotherapies in NSCLC. We report here the results of population-based PD-L1 testing using the 22C3 IHC pharmDx Assay (Agilent Technologies) in a large Canadian regional reference pathology laboratory.Testing was conducted reflexively on biopsies and resections for NSCLC during an 8-month period. Tumor proportion score (TPS) cutoffs for low and high expression were 1% and 50%, respectively.Altogether, 2031 PD-L1 tests were performed on specimens from 1795 patients, with 107 inconclusive results (5.3%). Excluding cases with inconclusive/missing data, proportions for the remaining 1713 patients were 41.6% for TPS less than 1%, 28.6% for TPS 1% to 49%, and 29.8% for TPS greater than or equal to 50%. Higher PD-L1 expression rates were noted in EGFR wild-type versus mutant tumors (p0.001), squamous versus adenocarcinoma (p0.001), and metastatic versus primary tumors (p0.001). PD-L1 among 103 patients with paired biopsy and resection specimens revealed moderate concordance (κ = 0.67). A total of 52% (25 of 48) of biopsies with TPS less than 1% had TPS greater than 1% in resection, whereas 84.6% (22 of 26) of biopsies with TPS greater than or equal to 50% were concordant in resected tumors. Discordance rates between biopsy and resection were 71.4% for biopsies with less than 8 mmIntratumoral heterogeneity of PD-L1 expression may result in misclassification of PD-L1 status in a substantial proportion of PD-L1-negative small biopsy samples. Biopsy of metastatic site may increase proportion of patients with high PD-L1 expression.

Introduction A grading system for pulmonary adenocarcinoma has not been established. The International Association for the Study of Lung Cancer pathology panel evaluated a set of histologic criteria associated with prognosis aimed at establishing a grading system for invasive pulmonary adenocarcinoma. Methods A multi-institutional study involving multiple cohorts of invasive pulmonary adenocarcinomas was conducted. A cohort of 284 stage I pulmonary adenocarcinomas was used as a training set to identify histologic features associated with patient outcomes (recurrence-free survival [RFS] and overall survival [OS]). Receiver operating characteristic curve analysis was used to select the best model, which was validated (n = 212) and tested (n = 300, including stage I–III) in independent cohorts. Reproducibility of the model was assessed using kappa statistics. Results The best model (area under the receiver operating characteristic curve [AUC] = 0.749 for RFS and 0.787 for OS) was composed of a combination of predominant plus high-grade histologic pattern with a cutoff of 20% for the latter. The model consists of the following: grade 1, lepidic predominant tumor; grade 2, acinar or papillary predominant tumor, both with no or less than 20% of high-grade patterns; and grade 3, any tumor with 20% or more of high-grade patterns (solid, micropapillary, or complex gland). Similar results were seen in the validation (AUC = 0.732 for RFS and 0.787 for OS) and test cohorts (AUC = 0.690 for RFS and 0.743 for OS), confirming the predictive value of the model. Interobserver reproducibility revealed good agreement (k = 0.617). Conclusions A grading system based on the predominant and high-grade patterns is practical and prognostic for invasive pulmonary adenocarcinoma.

The application of ex vivo lung perfusion (EVLP) has significantly increased the successful clinical use of marginal donor lungs. While large animal EVLP models exist to test new strategies to improve organ repair, there is currently no rat EVLP model capable of maintaining long-term lung viability. Here, we describe a new rat EVLP model that addresses this need, while enabling the study of lung injury due to cold ischemic time (CIT). The technique involves perfusing and ventilating male Lewis rat donor lungs for 4 h before transplanting the left lung into a recipient rat and then evaluating lung function 2 h after reperfusion. To test injury within this model, lungs were divided into groups and exposed to different CITs (i.e., 20 min, 6 h, 12 h, 18 h and 24 h). Experiments involving the 24-h-CIT group were prematurely terminated due to the development of severe edema. For the other groups, no differences in the ratio of arterial oxygen partial pressure to fractional inspired oxygen ([Formula: see text]/[Formula: see text]) were observed during EVLP; however, lung compliance decreased over time in the 18-h group ( P = 0.012) and the [Formula: see text]/[Formula: see text] of the blood from the left pulmonary vein 2 h after transplantation was lower compared with 20-min-CIT group ( P = 0.0062). This new model maintained stable lung function during 4-h EVLP and after transplantation when exposed to up to 12 h of CIT.

Long-term outcomes after lung transplantation remain inferior to those of other solid organ groups. The significance of eosinophils detected on transbronchial biopsies (TBBx) after lung transplantation and their relationship to long-term outcomes remain unknown. A retrospective single-center cohort study was performed of patients transplanted between January 01, 2001, and July 31, 2018, who had at least 1 TBBx with evaluable parenchymal tissue. Multivariable Cox proportional hazard models were used to assess the associations between eosinophil detection and: all-cause mortality and Chronic Lung Allograft Dysfunction (CLAD). 8887 TBBx reports from 1440 patients were reviewed for the mention of eosinophils in the pathology report. 112 (7.8%) patients were identified with eosinophils on at least one TBBx. The median (95% CI) survival time for all patients was 8.28 (7.32-9.31) years. Multivariable analysis, adjusted for clinical variables known to affect post-transplant outcomes, showed that the detection of eosinophils was independently associated with an increased risk of death (HR 1.51, 95% CI 1.24-1.85, p < 0.01) and CLAD (HR 1.35, 95% CI 1.07-1.70, P = 0.01). Eosinophils detected in TBBx are associated with an increased risk of CLAD and death. There may be benefit in specifically reporting the presence of eosinophils in TBBx reports and incorporating their presence in clinical decision-making.

Introduction The programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) assay is used to select patients for first or second-line pembrolizumab monotherapy in NSCLC. The PD-L1 IHC 22C3 pharmDx assay requires an Autostainer Link 48 instrument. Laboratories without this stainer have the option to develop a highly accurate 22C3 IHC laboratory-developed test (LDT) on other instruments. The Canadian 22C3 IHC LDT validation project was initiated to harmonize the quality of PD-L1 22C3 IHC LDT protocols across 20 Canadian pathology laboratories. Methods Centrally optimized 22C3 LDT protocols were distributed to participating laboratories. The LDT results were assessed against results using reference PD-L1 IHC 22C3 pharmDx. Analytical sensitivity and specificity were assessed using cell lines with varying PD-L1 expression levels (phase 1) and IHC critical assay performance controls (phase 2B). Diagnostic sensitivity and specificity were assessed using whole sections of 50 NSCLC cases (phase 2A) and tissue microarrays with an additional 50 NSCLC cases (phase 2C). Results In phase 1, 80% of participants reached acceptance criteria for analytical performance in the first attempt with disseminated protocols. However, in phase 2A, only 40% of participants reached the desired diagnostic accuracy for both 1% and 50% tumor proportion score cutoff. In phase 2B, further protocol modifications were conducted, which increased the number of successful laboratories to 75% in phase 2C. Conclusions It is possible to harmonize highly accurate 22C3 LDTs for both 1% and 50% tumor proportion score in NSCLC across many laboratories with different platforms. However, despite a centralized approach, diagnostic validation of predictive IHC LDTs can be challenging and not always successful.

Background Increasing evidence indicates that regulated necrosis plays a critical role during cell death caused by ischemia-reperfusion (IR) injury. Necroptosis is one form of regulated necrosis. Necrostatin-1 (Nec-1), an inhibitor of receptor-interacting protein kinase 1 (RIPK1), is known to reduce necroptosis. We investigated the effect of Nec-1 treatment on IR-induced lung injury in a rat lung transplant model. Methods Lewis rats were divided into 4 groups (n = 6 each): (1) Control (no treatment), (2) Donor treatment (D), (3) Recipient treatment (R), and (4) Donor plus Recipient treatment (D+R) groups. Donor lungs were flushed and preserved for 18 hours at 4oC before transplantation. Recipient animals underwent a left single lung transplant. After 2 hours of reperfusion, we assessed the physiologic function, cytokine expression, pathway activation, and the extent of necrosis. Results Pulmonary gas exchange in D+R group was significantly better than in the other 3 groups ( p = 0.003). Lung edema was significantly lower in the D+R group compared with the Control group (p = 0.006). The expression of interleukin-6 in lung tissue and plasma was significantly reduced in the D+R group compared with the Control group (p = 0.036). The percentage of necrotic cells in D+R group was significantly lower than in the Control and D groups (p = 0.01), indicating Nec-1inhibited regulated necrosis. Conclusions The administration of Nec-1 to both donor and recipient improved graft function after lung transplantation through the reduction of necroptosis. The inhibition of regulated necrosis appears to be a promising strategy to attenuate IR lung injury after lung transplantation.

Introduction Somatic mutations are becoming increasingly important biomarkers for treatment selection and outcome in patients with non-small-cell lung cancer (NSCLC). The role of multiple somatic mutations in early-stage NSCLC is unclear. Methods Tissue from 214 patients with resected NSCLC at the Princess Margaret Cancer Centre was analyzed by next-generation sequencing by Mi-SEQ or Sequenom multiplex platforms. Associations between mutation status, baseline patient characteristics and outcomes (disease-free survival (DFS) after surgical resection and overall survival (OS)) were investigated. Results Somatic mutations were identified in 184 patients with resected stage I-III NSCLC: None (n = 30), single (n = 101) and multiple (≥2, n = 83). Multiple mutations were significantly associated with younger age (p = 0.0006), female sex (p = 0.012), smoking status (p = 0.002) and adenocarcinoma histology (p = 0.0001).TP53, KRAS and EGFR were the most common mutations. TP53 mutation was the most frequent co-mutation occurring in 72% of patients with multiple mutations. In resected stage I-III patients, multiple mutations were significantly associated with worse DFS (HR = 2.56, p = 0.003) but not OS on univariate analysis. Patients with KRAS and EGFR mutations were also associated with shorter DFS (HR = 2.52, p = 0.016 and HR = 4.37, p = 0.001 respectively) but no OS difference. TP53 mutation was associated with both shorter DFS (HR = 2.21, p = 0.02) and OS (HR = 3.08, p = 0.02). In subgroup univariate analysis, poorer DFS was associated with multiple mutations (p = 0.0015), EGFR (HR = 3.14, p = 0.006), and TP53 (HR = 2.46, p = 0.018) in patients with stage I disease. Conclusion The presence of known somatic mutations is associated with worse DFS in resected NSCLC. The differences are both statistically significant and clinically relevant. The presence of EGFR, KRAS and TP53 mutations was also associated with adverse outcomes. Larger datasets are required to validate whether mutational status is an independent prognostic factor in early stage NSCLC.

Background Molecular testing in advanced lung cancer is standard in guiding treatment selection. However, population-wide implementation of testing remains a challenge. We developed a knowledge translation intervention to improve understanding among diagnostic specialists about molecular testing and appropriate diagnostic sampling in lung cancer. Methods Specialty-specific education programs were developed from existing literature and input from Canadian leaders in lung pathology, respirology, interventional radiology, thoracic surgery, radiation oncology, and medical oncology. The programs, including key messages, review of current data, existing guidelines, group discussion, and participant feedback, were administered at provincial and national specialty meetings. Participant knowledge was assessed before and after the intervention by using anonymous questionnaires. Molecular ( EGFR ) testing rates in Ontario were also evaluated before and after the intervention period. Results Ten programs were administered to diagnostic specialists, including respirologists, pathologists, thoracic surgeons, radiologists, radiation oncologists, and medical oncologists, with completion of 255 preintervention and 219 postintervention surveys. At baseline, 30% were unsure of tissue handling methods for molecular testing, 20% chose an incorrect technique, and half were unfamiliar with how to initiate testing. After intervention, specialist knowledge improved regarding tissue handling and appropriate fixation techniques and uncertainty decreased from 30% to 2% ( p EGFR ) testing requests in Ontario was observed over the intervention period ( p = 0.0032). Conclusions Significant knowledge gaps exist among diagnostic specialists regarding molecular testing and targeted therapy in lung cancer. This initiative significantly improved understanding of the importance and methods of successful molecular testing and correlated with increased testing rates.

Background Ex-vivo lung perfusion (EVLP), a technique for donor lung assessment, also represents a platform for donor lung repair and immunomodulation. α 1 -Anti-trypsin (A1AT), a medication used to treat emphysema in A1AT-deficient patients, has anti-inflammatory properties and has been shown to attenuate ischemia–reperfusion injury in rat and pig lung transplants . The objective of this study was to determine whether administration of A1AT during EVLP can improve donor lung quality after prolonged hypothermic preservation. Methods Pig donor lungs were retrieved, preserved at 4°C for 24 hours, and then subjected to normothermic EVLP for 12 hours using the Toronto protocol. The treatment group (n = 6) received 3 mg/ml A1AT (Zemaira) in the EVLP perfusate, acellular Steen solution. The control group (n = 6) was perfused with Steen solution only. Physiologic functions and gas exchange were measured hourly. Pulmonary edema, lung injury, apoptosis and inflammatory mediators were evaluated in lung tissues and perfusate. Results A1AT treatment significantly reduced pulmonary arterial pressure , pulmonary vascular resistance and airway pressure changes from the baseline when compared with controls. A1AT treatment significantly improved both dynamic and static pulmonary compliance , and change in partial pressure of oxygen (ΔPO 2 ) between the left atrium and the pulmonary artery. Furthermore, A1AT treatment also significantly reduced pulmonary edema (wet-to-dry ratio), pulmonary cell apoptosis and pro-inflammatory cytokine levels (interleukin-1α and -8) in the perfusate. Conclusion Treatment of 24-hour-preserved pig donor lungs with A1AT during EVLP resulted in improved physiologic function, reduced pulmonary edema and inflammation and decreased cell death. Our findings suggest that treatment of donor lungs during EVLP with A1AT is a promising strategy to attenuate early lung injury and improve donor lung function before lung transplantation.

// Grzegorz J. Korpanty 1 , Suzanne Kamel-Reid 2 , Melania Pintilie 1 , David M. Hwang 3 , Alona Zer 1 , Geoffrey Liu 1 , Natasha B. Leighl 1 , Ronald Feld 1 , Lillian L. Siu 1 , Philippe L. Bedard 1 , Ming-Sound Tsao 3 and Frances A. Shepherd 1 1 Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada 2 Laboratory Genetics, University Health Network, Toronto, ON, Canada 3 Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada Correspondence to: Grzegorz J. Korpanty, email: greg.korpanty@gmail.com Keywords: lung cancer; never smokers; targeted therapy Received: September 27, 2017 Accepted: March 23, 2018 Published: April 27, 2018 ABSTRACT Introduction: Lung cancer in never smokers represents a distinct epidemiological, clinical, and molecular entity. Results: Most 712 never smoking lung cancer patients were female (72%) with a median age at diagnosis of 62.2 years (18–94). Caucasians (46%), East Asians (42%), adenocarcinoma histology (87%) and presentation with metastatic disease at diagnosis (59%) were common. Of 515 patients with available archival tissue, the most common identified single mutations were EGFR (52.2%), followed by ALK (7.5%), KRAS (2.3%), TP53 (1.3%), ERBB2 (1%), BRAF (0.4%), PIK3CA (0.4%), SMAD4 (0.4%), CTNNB1 (0.2%), AKT1 (0.2%), and NRAS (0.2%); 8% tumors had multiple mutations, while 25.8% had none identified. Median overall survival (mOS) was 42.2 months (mo) for the entire cohort. Patients with mutations in their tumors had significantly better mOS (69.5 mo) when compared to those without (31.0 mo) (HR = 0.59; 95% CI: 0.44–0.79; p < 0.001). Earlier stage ( p < 0.001), adenocarcinoma histology ( p = 0.012), good performance status ( p < 0.001) and use of targeted therapy ( p < 0.001) were each independently associated with longer survival. Patients with ALK -translocation-positive tumours have significantly longer OS compared to those without any mutations ( p = 0.0029) and to those with other and null mutations ( p = 0.022). Conclusions: Lung cancer in never smokers represents a distinct clinical and molecular entity characterized by a high incidence of targetable mutations and long survival. Methods: We analyzed retrospectively the data from electronic patient records of never smokers diagnosed with lung cancer treated at the Princess Margaret Cancer Centre (Toronto) between 1988–2015 to characterize demographic and clinical features, pathology, molecular profile (using hotspot or targeted sequencing panels), treatment and survival.

Purpose Long-term survival after a lung transplantation (LTx) remains limited by Chronic Lung Allograft Dysfunction (CLAD). B-grade airway-centered acute rejection (AR) has been associated with subsequent CLAD. B-grade AR is considered a cause of airway epithelial damage, but the type of injury and specific epithelial cells at risk are poorly understood. We aimed to assess the detailed morphology of lung allograft epithelium during B-grade AR. Methods We retrospectively identified all transbronchial biopsies (TBBx) with airway-centered AR (B-grade ≥B1), collected 2-25 months after LTx from patients with a first bilateral LTx performed 2010-2015: 38 TBBx from 36 patients. 15 A0B0 TBBx from separate patients were matched to the ≥B1 TBBx according to time post LTx. Blinded semi-quantitative (scores 0-4) grading was performed of each airway using Hematoxylin & Eosin-stained sections to assess features of airway fibrosis and epithelial changes previously associated with CLAD. Immunofluorescence staining was performed using antibodies against club cell secretory protein (CCSP) and Muc-5AC. Results B-grade AR with concurrent ≥10% decline in lung function was associated with an increased risk of CLAD compared to stable B-grade AR and stable B0 TBBx (p=0.01) (figure 1A). Obliterative airway fibrosis (p=0.001) and epithelial flattening (p=0.0004) scores were higher in patients with ≥B1 compared to B0 (figure 1B). We found that epithelial hyperplasia increased over time after transplant (p=0.003) (figure 1C). The percentage of cells positive for CCSP and Muc-5AC was not different between ≥B1 and B0 TBBx (figure 1D), was not associated with time between LTx and TBBx, and did not correlate with our epithelial grading results. Conclusion The combination of B-grade AR and declining lung function is a predictor of CLAD. B-grade AR is associated with histologic obliterative airway fibrosis and epithelial flattening. Further analyses of specific epithelial cell types and changes during B-grade AR are ongoing.

BackgroundSurvival after lung transplantation (LTx) is hampered by uncontrolled inflammation and alloimmunity. Regulatory T-cells (Tregs) are being studied as a cellular therapy in solid organ transplantation. Whether these systemically administered Tregs can function at the appropriate location and time is an important concern. We hypothesised that in vitro-expanded recipient-derived Tregs can be delivered to donor lungs prior to LTx via ex vivo lung perfusion (EVLP), maintaining their immunomodulatory ability.MethodsIn a rat model, Wistar Kyoto (WKy) CD4+CD25high Tregs were expanded in vitro prior to EVLP. Expanded Tregs were administered to Fisher 344 (F344) donor lungs during EVLP; left lungs were transplanted into WKy recipients. Treg localisation and function post-transplant were assessed. In a proof-of-concept experiment, cryopreserved expanded human CD4+CD25+CD127low Tregs were thawed and injected into discarded human lungs during EVLP.ResultsRat Tregs entered the lung parenchyma and retained suppressive function. Expanded Tregs had no adverse effect on donor lung physiology during EVLP; lung water as measured by wet-to-dry weight ratio was reduced by Treg therapy. The administered cells remained in the graft at 3 days post-transplant where they reduced activation of intra-graft effector CD4+ T-cells; these effects were diminished by day 7. Human Tregs entered the lung parenchyma during EVLP where they expressed key immunoregulatory molecules (CTLA4+, 4-1BB+, CD39+ and CD15s+).ConclusionsPre-transplant Treg administration can inhibit alloimmunity within the lung allograft at early time points post-transplant. Our organ-directed approach has potential for clinical translation.

Summary Background Results from retrospective studies indicate that selecting individuals for low-dose CT lung cancer screening on the basis of a highly predictive risk model is superior to using criteria similar to those used in the National Lung Screening Trial (NLST; age, pack-year, and smoking quit-time). We designed the Pan-Canadian Early Detection of Lung Cancer (PanCan) study to assess the efficacy of a risk prediction model to select candidates for lung cancer screening, with the aim of determining whether this approach could better detect patients with early, potentially curable, lung cancer. Methods We did this single-arm, prospective study in eight centres across Canada. We recruited participants aged 50–75 years, who had smoked at some point in their life (ever-smokers), and who did not have a self-reported history of lung cancer. Participants had at least a 2% 6-year risk of lung cancer as estimated by the PanCan model, a precursor to the validated PLCOm2012 model. Risk variables in the model were age, smoking duration, pack-years, family history of lung cancer, education level, body-mass index, chest x-ray in the past 3 years, and history of chronic obstructive pulmonary disease. Individuals were screened with low-dose CT at baseline (T0), and at 1 (T1) and 4 (T4) years post-baseline. The primary outcome of the study was incidence of lung cancer. This study is registered with ClinicalTrials.gov, number NCT00751660. Findings 7059 queries came into the study coordinating centre and were screened for PanCan risk. 15 were duplicates, so 7044 participants were considered for enrolment. Between Sept 24, 2008, and Dec 17, 2010, we recruited and enrolled 2537 eligible ever-smokers. After a median follow-up of 5·5 years (IQR 3·2–6·1), 172 lung cancers were diagnosed in 164 individuals (cumulative incidence 0·065 [95% CI 0·055–0·075], incidence rate 138·1 per 10 000 person-years [117·8–160·9]). There were ten interval lung cancers (6% of lung cancers and 6% of individuals with cancer): one diagnosed between T0 and T1, and nine between T1 and T4. Cumulative incidence was significantly higher than that observed in NLST (4·0%; p Interpretation The PanCan model was effective in identifying individuals who were subsequently diagnosed with early, potentially curable, lung cancer. The incidence of cancers detected and the proportion of early stage cancers in the screened population was higher than observed in previous studies. This approach should be considered for adoption in lung cancer screening programmes. Funding Terry Fox Research Institute and Canadian Partnership Against Cancer.

Background Patients with lung cancer are at increased risk for venous thromboembolism (VTE), particularly those receiving chemotherapy. It is estimated that 8% to 15% of patients with advanced non–small-cell lung cancer (NSCLC) experience a VTE in the course of their disease. The incidence in patients with specific molecular subtypes of NSCLC is unknown. We undertook this review to determine the incidence of VTE in patients with ALK (anaplastic lymphoma kinase)-rearranged NSCLC. Patients and Methods We identified all patients with ALK -rearranged NSCLC diagnosed and/or treated at the Princess Margaret Cancer Centre (PM CC) in Canada between July 2012 and January 2015. Retrospective data were extracted from electronic medical records. We then included a validation cohort comprising all consecutive patients with ALK -rearranged NSCLC treated in 2 tertiary centers in Israel. Results Within the PM CC cohort, of 55 patients with ALK -rearranged NSCLC, at a median follow-up of 22 months, 23 (42%) experienced VTE. Patients with VTE were more likely to be white ( P = .006). The occurrence of VTE was associated with a trend toward worse prognosis (overall survival hazard ratio = 2.88, P = .059). Within the validation cohort (n = 43), the VTE rate was 28% at a median follow-up of 13 months. Combining the cohorts (n = 98), the VTE rate was 36%. Patients with VTE were younger (age 52 vs. 58 years, P = .04) and had a worse Eastern Cooperative Oncology Group performance status ( P = .04). VTE was associated with shorter overall survival (hazard ratio = 5.71, P = .01). Conclusion The rate of VTE in our ALK -rearranged cohort was 3- to 5-fold higher than previously reported for the general NSCLC population. This warrants confirmation in larger cohorts.

Purpose Mouse orthotopic single lung transplantation (LTx) is a vital scientific model to explore lung transplantation (LTx) immunology. Minor alloantigen-mismatched LTx using C57BL/10 (B10, H-2b) to C57BL/6 (B6, H-2b) has mild acute rejection and sometimes chronic fibrosis, mimicking human LTx where alloimmunity is dampened by immunosuppressants. We have observed variations in allograft histology depending on individual microsurgeons’ experience. The purpose of this study was to compare pathological variations between individual surgeons and over time. Methods We performed a retrospective review of B10 to B6 LTx performed in our lab and sacrificed 28 days after LTx. LTx with an experimental intervention (e.g., usage of immunomodulatory agents or knock-out mice) were excluded. All pathological grading was performed in a blinded manner using HE B grade, Pl0.0001; % parenchymal fibrosis, P=0.1164; peri-airway fibrosis, Pl0.0001; obliterative airway fibrosis, Pl0.0001). Intra-surgeon chronological changes in pathological scores were statistically significant for A grade, B grade, and % parenchymal fibrosis (P=0.0024, P=0.0416, P=0.0097, respectively). Conclusion Mouse single lung transplantation is one of the most technically challenging animal models. It remains a powerful model to study underlying mechanisms of LTx biology. However, the significant learning curve and surgeon-dependent variability in histopathological findings need to be taken into account when designing experimental protocols.

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.

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.

Acute cellular rejection (ACR) is a significant risk factor for chronic lung allograft dysfunction (CLAD). Although clinically manifest and higher grade (≥A2) ACR is generally treated with augmented immunosuppression, management of minimal (grade A1) ACR remains controversial. In our program, patients with subclinical and spirometrically stable A1 rejection (StA1R) are routinely not treated with augmented immunosuppression. We hypothesized that an untreated first StA1R does not increase the risk of CLAD or death compared to episodes of spirometrically stable no ACR (StNAR). The cohort was drawn from all consecutive adult, first, bilateral lung transplantations performed between 1999 and 2017. Biopsies obtained in the first-year posttransplant were paired with (forced expiratory volume in 1 second FEV1 ). The first occurrence of StA1R was compared to a time-matched StNAR. The risk of CLAD or death was assessed using univariable and multivariable Cox proportional hazards models. The analyses demonstrated no significant difference in risk of CLAD or death in patients with a first StA1R compared to StNAR. This largest study to date shows that, in clinically stable patients, an untreated first A1 ACR in the first-year posttransplant is not significantly associated with an increased risk for CLAD or death. Watchful-waiting approach may be an acceptable tactic for stable A1 episodes in lung transplant recipients.

Chronic lung allograft dysfunction (CLAD) limits long-term survival after lung transplant (LT). Ischemia-reperfusion injury (IRI) promotes chronic rejection (CR) and CLAD, but the underlying mechanisms are not well understood. To examine mechanisms linking IRI to CR, a mouse orthotopic LT model using a minor alloantigen strain mismatch (C57BL/10 [B10, H-2b ] → C57BL/6 [B6, H-2b ]) and isograft controls (B6→B6) was used with antecedent minimal or prolonged graft storage. The latter resulted in IRI with subsequent airway and parenchymal fibrosis in prolonged storage allografts but not isografts. This pattern of CR after IRI was associated with the formation of B cell-rich tertiary lymphoid organs within the grafts and circulating autoantibodies. These processes were attenuated by B cell depletion, despite preservation of allograft T cell content. Our observations suggest that IRI may promote B cell recruitment that drives CR after LT. These observations have implications for the mechanisms leading to CLAD after LT.

The Lung session of the 2017 14th Banff Foundation for Allograft Pathology Conference, Barcelona focused on the multiple aspects of antibody-mediated rejection (AMR) in lung transplantation. Multidimensional approaches for AMR diagnosis, including classification, histological and immunohistochemical analysis, and donor- specific antibody (DSA) characterization with their current strengths and limitations were reviewed in view of recent research. The group also discussed the role of tissue gene expression analysis in the context of unmet needs in lung transplantation. The current best practice for monitoring of AMR and the therapeutic approach are summarized and highlighted in this report. The working group reached consensus of the major gaps in current knowledge and focused on the unanswered questions regarding pulmonary AMR. An important outcome of the meeting was agreement on the need for future collaborative research projects to address these gaps in the field of lung transplantation.

Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomics study of 111 B. multivorans sputum isolates from one CF patient through three stages of infection including an early incident isolate, deep sampling of a one-year period of chronic infection occurring weeks before a lung transplant, and deep sampling of a post-transplant infection. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found the incident isolate was basally related to the rest of the strains and more susceptible to antibiotics from three classes (β-lactams, aminoglycosides, quinolones). The chronic infection isolates diversified into multiple, distinct genetic lineages and showed reduced antimicrobial susceptibility to the same antibiotics. The post-transplant reinfection isolates derived from the same source as the incident isolate and were genetically distinct from the chronic isolates. They also had a level of susceptibility in between that of the incident and chronic isolates. We identified numerous examples of potential parallel pathoadaptation, in which multiple mutations were found in the same locus or even codon. The set of parallel pathoadaptive loci was enriched for functions associated with virulence and resistance. Our GWAS analysis identified statistical associations between a polymorphism in the ampD locus with resistance to β-lactams, and polymorphisms in an araC transcriptional regulator and an outer membrane porin with resistance to both aminoglycosides and quinolones. Additionally, these three loci were independently mutated four, three and two times, respectively, providing further support for parallel pathoadaptation. Finally, we identified a minimum of 14 recombination events, and observed that loci carrying putative parallel pathoadaptations and polymorphisms statistically associated with β-lactam resistance were over-represented in these recombinogenic regions., Author summary Cystic fibrosis (CF) is the most common lethal genetic disorder affecting individuals of European descent. Most CF patients die at a young age due to chronic lung infections. Among the organisms involved in these infections are bacteria from the Burkholderia cepacia complex (BCC), which are strongly associated with poor clinical prognosis. This study examines how the most prevalent BCC species among CF patients, B. multivorans, evolves within a single CF patient by studying the first B. multivorans isolate recovered from the patient, one hundred isolates recovered over a one year period during the chronic infection phase, and an additional ten isolates recovered after the reinfection of the transplanted lungs. We found that B. multivorans diversify phenotypically and genetically within the CF lung over the course of the infection, and evolves into a complex population during the chronic infection phase. We found that isolates collected from the post-transplant reinfection were more closely related to descendants of the original isolate rather than those recovered in the chronic infection. We identify genetic variants statistically associated with resistance to the antibiotics, and showed that some of these variants were found in regions that show patterns of recombination (genetic exchange) between strains. We also found that genes which were mutated multiple times during overall infection were more likely to be found in regions showing signals consistent with recombination. The presence of multiple independent mutations in a gene is a very strong signal that the gene helps bacteria adapt to their environment. Overall, this study provides insight into how pathogens adapt to the host during long-term infections, specific genes associated with antibiotic resistance, and the origin of new and recurrent infections.