Implementation of Liquid Biopsy in Non-Small-Cell Lung Cancer: An Ontario Perspective.

Lung cancer is the leading cause of cancer-related deaths in Canada, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. Timely access to comprehensive molecular profiling is critical for selecting biomarker-matched targeted therapies, which lead to improved outcomes in advanced NSCLC. Tissue biopsy samples are the gold standard for molecular profiling; however, several challenges can prevent timely and complete molecular profiling from being performed, causing delays in treatment or suboptimal therapy selection. Liquid biopsy offers a minimally invasive method for molecular profiling by analyzing circulating tumour DNA (ctDNA) and RNA (cfRNA) in plasma, potentially overcoming these barriers. This paper discusses the outcomes of a multidisciplinary working group in Ontario, which proposed three eligibility criteria for liquid biopsy reimbursement: (1) insufficient tissue for complete testing or failed tissue biomarker testing; (2) suspected advanced NSCLC where tissue biopsy is not feasible; and (3) high-risk patients who may deteriorate before tissue results are available. The group also addressed considerations for assay selection, implementation, and economic impact. These discussions aim to inform reimbursement and implementation strategies for liquid biopsy in Ontario's public healthcare system, recognizing the need for ongoing evaluation as technology and evidence evolve.

Data set for the reporting of lung cancer: recommendations from the International Collaboration on Cancer Reporting (ICCR).

Lung cancer is the leading cause of cancer related deaths worldwide, although some patients with early-stage disease can be cured with surgical resection. Standardised reporting of all clinically relevant pathological parameters is essential for best patient care and is also important for ongoing data collection and refinement of important pathological features that impact patient prognosis, staging and clinical care. Using the established International Collaboration on Cancer Reporting (ICCR) procedure, a representative international expert panel of nine lung pathologists as well as an oncologist was convened. Essential core elements and suggested non-core elements were identified for inclusion in the resected lung cancer pathology data set based on predetermined levels of evidence as well as consensus expert opinion. A lung cancer histopathology reporting guide was developed that includes relevant clinical, macroscopic, microscopic and ancillary testing. Critical review and discussion of current evidence was incorporated into the new data set including changes from the 2021 World Health Organisation (WHO) Classification of Thoracic Tumours, fifth edition, new requirements for grading invasive non-mucinous adenocarcinomas, assessment of response to neoadjuvant therapy and requirements for molecular testing in early-stage resected lung carcinomas. This ICCR data set represents incorporation of all relevant parameters for histology reporting of lung cancer resection specimens. Routine use of this data set is recommended for all pathology reporting of resected lung cancer and it is freely available worldwide on the ICCR website (https://www.iccr-cancer.org/datasets/published-datasets/). Widespread implementation will help to ensure consistent and comprehensive pathology reporting and data collection essential for lung cancer patient care, clinical trials and other research.

Lung Transplant Immunomodulation with Genetically Engineered Mesenchymal Stromal Cells-Therapeutic Window for Interleukin-10.

Lung transplantation results are compromised by ischemia-reperfusion injury and alloimmune responses. Ex vivo lung perfusion (EVLP) is used to assess marginal donor lungs before transplantation but is also an excellent platform to apply novel therapeutics. We investigated donor lung immunomodulation using genetically engineered mesenchymal stromal cells with augmented production of human anti-inflammatory hIL-10 (MSCsIL-10). Pig lungs were placed on EVLP for 6 h and randomized to control (n = 7), intravascular delivery of 20 × 106 (n = 5, low dose) or 40 × 106 human MSCs IL-10 (n = 6, high dose). Subsequently, single-lung transplantation was performed, and recipient pigs were monitored for 3 days. hIL-10 secretion was measured during EVLP and after transplantation, and immunological effects were assessed by cytokine profile, T and myeloid cell characterization and mixed lymphocyte reaction. MSCIL-10 therapy rapidly increased hIL-10 during EVLP and resulted in transient hIL-10 elevation after lung transplantation. MSCIL-10 delivery did not affect lung function but was associated with dose-related immunomodulatory effects, with the low dose resulting in a beneficial decrease in apoptosis and lower macrophage activation, but the high MSCIL-10 dose resulting in inflammation and cytotoxic CD8+ T cell activation. MSCIL-10 therapy during EVLP results in a rapid and transient perioperative hIL-10 increase and has a therapeutic window for its immunomodulatory effects.

Programmed Death Ligand-1 and Tumor Mutation Burden Testing of Patients With Lung Cancer for Selection of Immune Checkpoint Inhibitor Therapies: Guideline From the College of American Pathologists, Association for Molecular Pathology, International Association for the Study of Lung Cancer, Pulmonary Pathology Society, and LUNGevity Foundation.

CONTEXT.­: Rapid advancements in the understanding and manipulation of tumor-immune interactions have led to the approval of immune therapies for patients with non-small cell lung cancer. Certain immune checkpoint inhibitor therapies require the use of companion diagnostics, but methodologic variability has led to uncertainty around test selection and implementation in practice. OBJECTIVE.­: To develop evidence-based guideline recommendations for the testing of immunotherapy/immunomodulatory biomarkers, including programmed death ligand-1 (PD-L1) and tumor mutation burden (TMB), in patients with lung cancer. DESIGN.­: The College of American Pathologists convened a panel of experts in non-small cell lung cancer and biomarker testing to develop evidence-based recommendations in accordance with the standards for trustworthy clinical practice guidelines established by the National Academy of Medicine. A systematic literature review was conducted to address 8 key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were created from the available evidence, certainty of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS.­: Six recommendation statements were developed. CONCLUSIONS.­: This guideline summarizes the current understanding and hurdles associated with the use of PD-L1 expression and TMB testing for immune checkpoint inhibitor therapy selection in patients with advanced non-small cell lung cancer and presents evidence-based recommendations for PD-L1 and TMB testing in the clinical setting.

Utility of molecular markers in predicting local control specific to lung cancer spine metastases treated with stereotactic body radiotherapy.

BACKGROUND AND PURPOSE: We report outcomes following spine stereotactic body radiotherapy (SBRT) in metastatic non-small cell lung cancer (NSCLC) and the significance of programmed death-ligand 1 (PD-L1) status, epidermal growth factor receptor (EGFR) mutation and timing of immune check point inhibitors (ICI) on local failure (LF). MATERIALS AND METHODS: 165 patients and 389 spinal segments were retrospectively reviewed from 2009 to 2021. Baseline patient characteristics, treatment and outcomes were abstracted. Primary endpoint was LF and secondary, overall survival (OS) and vertebral compression fracture (VCF). Multivariable analysis (MVA) evaluated factors predictive of LF and VCF. RESULTS: The median follow-up and OS were: 13.0 months (range, 0.5-95.3 months) and 18.4 months (95% CI 11.4-24.6). 52.1% were male and 76.4% had adenocarcinoma. Of the 389 segments, 30.3% harboured an EGFR mutation and 17.0% were PD-L1 ≥ 50%. The 24 months LF rate in PD-L1 ≥ 50% vs PD-L1 < 50% was 10.7% vs. 38.0%, and in EGFR-positive vs. negative was 18.1% vs. 30.0%. On MVA, PD-L1 status of ≥ 50% (HR 0.32, 95% CI 0.15-0.69, p = 0.004) significantly predicted for lower LF compared to PD-L1 < 50%. Lower LF trend was seen with ICI administration peri and post SBRT (HR 0.41, 95% CI 0.16-1.05, p = 0.062). On MVA, polymetastatic disease (HR 3.28, 95% CI 1.84-5.85, p < 0.0001) and ECOG ≥ 2 (HR 1.87, 95% CI 1.16-3.02, p = 0.011) significantly predicted for worse OS and absence of baseline VCF predicted for lower VCF rate (HR 0.20, 95% CI 0.10-0.39, p < 0.0001). CONCLUSION: We report a significant association of PD-L1 ≥ 50% status on improved LC rates from spine SBRT in NSCLC patients.

A New Cloud-Native Tool for Pharmacogenetic Analysis.

BACKGROUND: The advancement of next-generation sequencing (NGS) technologies provides opportunities for large-scale Pharmacogenetic (PGx) studies and pre-emptive PGx testing to cover a wide range of genotypes present in diverse populations. However, NGS-based PGx testing is limited by the lack of comprehensive computational tools to support genetic data analysis and clinical decisions. METHODS: Bioinformatics utilities specialized for human genomics and the latest cloud-based technologies were used to develop a bioinformatics pipeline for analyzing the genomic sequence data and reporting PGx genotypes. A database was created and integrated in the pipeline for filtering the actionable PGx variants and clinical interpretations. Strict quality verification procedures were conducted on variant calls with the whole genome sequencing (WGS) dataset of the 1000 Genomes Project (G1K). The accuracy of PGx allele identification was validated using the WGS dataset of the Pharmacogenetics Reference Materials from the Centers for Disease Control and Prevention (CDC). RESULTS: The newly created bioinformatics pipeline, Pgxtools, can analyze genomic sequence data, identify actionable variants in 13 PGx relevant genes, and generate reports annotated with specific interpretations and recommendations based on clinical practice guidelines. Verified with two independent methods, we have found that Pgxtools consistently identifies variants more accurately than the results in the G1K dataset on GRCh37 and GRCh38. CONCLUSIONS: Pgxtools provides an integrated workflow for large-scale genomic data analysis and PGx clinical decision support. Implemented with cloud-native technologies, it is highly portable in a wide variety of environments from a single laptop to High-Performance Computing (HPC) clusters and cloud platforms for different production scales and requirements.

Referred molecular testing as a barrier to optimal treatment decision making in metastatic non-small cell lung cancer: Experience at a tertiary academic institution in Canada.

BACKGROUND: Molecular testing is critical to guiding treatment approaches in patients with metastatic non-small cell lung cancer (mNSCLC), with testing delays adversely impacting the timeliness of treatment decisions. Here, we aimed to evaluate 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 mNSCLC 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, p < 0.0001), driven largely by decreased internal handling and specimen transit times (2 days vs. 3 days, p < 0.0001) and laboratory turnaround times (TAT, 3 days vs. 8 days, p < 0.0001), with 96% of in-house cases meeting the international guideline of a ≤ 10-day intra-laboratory TAT (vs. 74% send-out, p < 0.001). Eighty-eight percent of patients with in-house testing had results available at their first oncology consultation (vs. 52% send-out, p < 0.0001), and all patients with in-house testing had results available at the time of treatment decision (vs. 86% send-out, p = 0.57). CONCLUSION: Our results demonstrate the advantages of in-house biomarker testing for mNSCLC at a tertiary oncology center. Incorporation of in-house testing may reduce barriers to offering personalized medicine by improving the time to optimal systemic therapy decision.

Developing DPYD Genotyping Method for Personalized Fluoropyrimidines Therapy.

BACKGROUND: Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy. METHODS: A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129-5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing. RESULTS: From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129-5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129-5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results. CONCLUSIONS: A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.

Canadian Multicentric Pan-TRK (CANTRK) Immunohistochemistry Harmonization Study.

Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.

Donor IL-17 receptor A regulates LPS-potentiated acute and chronic murine lung allograft rejection.

Chronic lung allograft dysfunction (CLAD) is a major complication after lung transplantation that results from a complex interplay of innate inflammatory and alloimmune factors, culminating in parenchymal and/or obliterative airway fibrosis. Excessive IL-17A signaling and chronic inflammation have been recognized as key factors in these pathological processes. Herein, we developed a model of repeated airway inflammation in mouse minor alloantigen-mismatched single-lung transplantation. Repeated intratracheal LPS instillations augmented pulmonary IL-17A expression. LPS also increased acute rejection, airway epithelial damage, and obliterative airway fibrosis, similar to human explanted lung allografts with antecedent episodes of airway infection. We then investigated the role of donor and recipient IL-17 receptor A (IL-17RA) in this context. Donor IL-17RA deficiency significantly attenuated acute rejection and CLAD features, whereas recipient IL-17RA deficiency only slightly reduced airway obliteration in LPS allografts. IL-17RA immunofluorescence positive staining was greater in human CLAD lungs compared with control human lung specimens, with localization to fibroblasts and myofibroblasts, which was also seen in mouse LPS allografts. Taken together, repeated airway inflammation after lung transplantation caused local airway epithelial damage, with persistent elevation of IL-17A and IL-17RA expression and particular involvement of IL-17RA on donor structural cells in development of fibrosis.

Predictive modeling of antibiotic eradication therapy success for new-onset Pseudomonas aeruginosa pulmonary infections in children with cystic fibrosis.

Chronic Pseudomonas 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 clear 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 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.

Ultrasound-guided transfection of claudin-5 improves lung endothelial barrier function in lung injury without impairing innate immunity.

In acute lung injury, the lung endothelial barrier is compromised. Loss of endothelial barrier integrity occurs in association with decreased levels of the tight junction protein claudin-5. Restoration of their levels by gene transfection may improve the vascular barrier, but how to limit transfection solely to regions of the lung that are injured is unknown. We hypothesized that thoracic ultrasound in combination with intravenous microbubbles (USMBs) could be used to achieve regional gene transfection in injured lung regions and improve endothelial barrier function. Since air blocks ultrasound energy, insonation of the lung is only achieved in areas of lung injury (edema and atelectasis); healthy lung is spared. Cavitation of the microbubbles achieves local tissue transfection. Here we demonstrate successful USMB-mediated gene transfection in the injured lungs of mice. After thoracic insonation, transfection was confined to the lung and only occurred in the setting of injured (but not healthy) lung. In a mouse model of acute lung injury, we observed downregulation of endogenous claudin-5 and an acute improvement in lung vascular leakage and in oxygenation after claudin-5 overexpression by transfection. The improvement occurred without any impairment of the immune response as measured by pathogen clearance, alveolar cytokines, and lung histology. In conclusion, USMB-mediated transfection targets injured lung regions and is a novel approach to the treatment of lung injury.NEW & NOTEWORTHY Acute respiratory distress syndrome is characterized by spatial heterogeneity, with severely injured lung regions adjacent to relatively normal areas. This makes targeting treatment to the injured regions difficult. Here we use thoracic ultrasound and intravenous microbubbles (USMBs) to direct gene transfection specifically to injured lung regions. Transfection of the tight junction protein claudin-5 improved oxygenation and decreased vascular leakage without impairing innate immunity. These findings suggest that USMB is a novel treatment for ARDS.

Prognostic implications of and clinical risk factors for acute lung injury and organizing pneumonia after lung transplantation: Data from a multicenter prospective cohort study.

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.

EGFR Targeting TKI-Related Skin Toxicities in a Patient with Darker Skin: A Case Report.

Epidermal growth factor receptor (EGFR) targeting tyrosine kinase inhibitors (TKIs) can result in significant skin toxicities that may impact patients' quality of life. While these skin reactions are well documented in patients with lighter skin, there is a paucity of literature and images to guide clinicians in their assessment in patients with darker skin tones. Given that dermatological reactions in patients with darker skin are not well represented, this can result in the undertreatment or mistreatment of these otherwise common toxicities. Herein, we present a case of a female patient with a darker skin tone with metastatic non-small cell lung carcinoma (NSCLC) with EGFR-TKI-related skin toxicity and her clinical course.

CCTG BR34: A Randomized Phase 2 Trial of Durvalumab and Tremelimumab With or Without Platinum-Based Chemotherapy in Patients With Metastatic NSCLC.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Evaluation of digital dispense-assisted broth microdilution antimicrobial susceptibility testing for Pseudomonas aeruginosa isolates.

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 (ß-lactams, ß-lactam/ß-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.

Prevalence and Heterogeneity of PD-L1 Expression by 22C3 Assay in Routine Population-Based and Reflexive Clinical Testing in Lung Cancer.

INTRODUCTION: 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. METHODS: 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. RESULTS: 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 (p < 0.001), squamous versus adenocarcinoma (p < 0.001), and metastatic versus primary tumors (p < 0.001). 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 mm2 total area, compared with 33.3% for biopsies with greater than or equal to 8 mm2 area (p < 0.026). Concordance among 27 patients with paired primary lung and metastatic tumor biopsies revealed only weak concordance (κ = 0.48). CONCLUSIONS: Intratumoral 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.

Eosinophils in transbronchial biopsies: a predictor of chronic lung allograft dysfunction and reduced survival after lung transplantation - a retrospective single-center cohort study.

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.

Ecological Succession of Polymicrobial Communities in the Cystic Fibrosis Airways.

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.

Canadian Multicenter Project on Standardization of Programmed Death-Ligand 1 Immunohistochemistry 22C3 Laboratory-Developed Tests for Pembrolizumab Therapy in NSCLC.

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.