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.

Genistein and Procyanidin B2 Reduce Carcinogen-Induced Reactive Oxygen Species and DNA Damage through the Activation of Nrf2/ARE Cell Signaling in Bronchial Epithelial Cells In Vitro.

Cancer is one of the leading causes of death worldwide. Chemotherapy and radiation therapy are currently providing the basis for cancer therapies, although both are associated with significant side effects. Thus, cancer prevention through dietary modifications has been receiving growing interest. The potential of selected flavonoids in reducing carcinogen-induced reactive oxygen species (ROS) and DNA damage through the activation of nuclear factor erythroid 2 p45 (NF-E2)-related factor (Nrf2)/antioxidant response element (ARE) pathway was studied in vitro. Dose-dependent effects of pre-incubated flavonoids on pro-carcinogen 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanone (NNKAc)-induced ROS and DNA damage in human bronchial epithelial cells were studied in comparison to non-flavonoids. The most effective flavonoids were assessed for the activation of Nrf2/ARE pathway. Genistein, procyanidin B2 (PCB2), and quercetin significantly suppressed the NNKAc-induced ROS and DNA damage. Quercetin significantly upregulated the phosphorylated protein kinase B/Akt. PCB2 significantly upregulated the activation of Nrf2 and Akt through phosphorylation. Genistein and PCB2 significantly upregulated the phospho-Nrf2 nuclear translocation and catalase activity. In summary, genistein and PCB2 reduced the NNKAc-induced ROS and DNA damage through the activation of Nrf2. Further studies are required to understand the role of dietary flavonoids on the regulation of the Nrf2/ARE pathway in relation to carcinogenesis.

"Interchangeability" of PD-L1 immunohistochemistry assays: a meta-analysis of diagnostic accuracy.

Different clones, protocol conditions, instruments, and scoring/readout methods may pose challenges in introducing different PD-L1 assays for immunotherapy. The diagnostic accuracy of using different PD-L1 assays interchangeably for various purposes is unknown. The primary objective of this meta-analysis was to address PD-L1 assay interchangeability based on assay diagnostic accuracy for established clinical uses/purposes. A systematic search of the MEDLINE database using PubMed platform was conducted using "PD-L1" as a search term for 01/01/2015 to 31/08/2018, with limitations "English" and "human". 2,515 abstracts were reviewed to select for original contributions only. 57 studies on comparison of two or more PD-L1 assays were fully reviewed. 22 publications were selected for meta-analysis. Additional data were requested from authors of 20/22 studies in order to enable the meta-analysis. Modified GRADE and QUADAS-2 criteria were used for grading published evidence and designing data abstraction templates for extraction by reviewers. PRISMA was used to guide reporting of systematic review and meta-analysis and STARD 2015 for reporting diagnostic accuracy study. CLSI EP12-A2 was used to guide test comparisons. Data were pooled using random-effects model. The main outcome measure was diagnostic accuracy of various PD-L1 assays. The 22 included studies provided 376 2×2 contingency tables for analyses. Results of our study suggest that, when the testing laboratory is not able to use an Food and Drug Administration-approved companion diagnostic(s) for PD-L1 assessment for its specific clinical purpose(s), it is better to develop a properly validated laboratory developed test for the same purpose(s) as the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic, than to replace the original PD-L1 Food and Drug Administration-approved immunohistochemistry companion diagnostic with a another PD-L1 Food and Drug Administration-approved companion diagnostic that was developed for a different purpose.

RTN4 Knockdown Dysregulates the AKT Pathway, Destabilizes the Cytoskeleton, and Enhances Paclitaxel-Induced Cytotoxicity in Cancers.

Reticulon-4 (RTN4), commonly known as a neurite outgrowth inhibitor (Nogo), is emerging as an important player in human cancers. Clinically, we found lower RTN4 expression in patient-derived tumors was associated with significantly better survival in lung, breast, cervical, and renal cancer patients. To identify the role of RTN4 in cancer biology, we performed mass spectrometry-based quantitative proteomic analysis on cancer cells following RTN4 knockdown and found its link with pro-survival as well as cytoskeleton-related processes. Subsequent mechanistic investigations revealed that RTN4 regulates lipid homeostasis, AKT signaling, and cytoskeleton modulation. In particular, downregulation of RTN4 reduced sphingomyelin synthesis and impaired plasma membrane localization of AKT, wherein AKT phosphorylation, involved in many cancers, was significantly reduced without any comparable effect on AKT-related upstream kinases, in a sphingolipid-dependent manner. Furthermore, knockdown of RTN4 retarded proliferation of cancer cells in vitro as well as tumor xenografts in mice. Finally, RTN4 knockdown affected tubulin stability and promoted higher cytotoxic effects with chemotherapeutic paclitaxel in cancer cells both in vitro and in vivo. In summary, RTN4 is involved in carcinogenesis and represents a molecular candidate that may be targeted to achieve desired antitumor effects in clinics.

Dirhamnolipid Produced by the Pathogenic Fungus Colletotrichum gloeosporioides BWH-1 and Its Herbicidal Activity.

Fungal phytotoxins used as ecofriendly bioherbicides are becoming efficient alternatives to chemical herbicides for sustainable weed management. Previous study found that cultures of the pathogenic fungus Colletotrichum gloeosporioides BWH-1 showed phytotoxic activity. This study further isolated the major phytotoxin from cultures of the strain BWH-1 using bioactivity-guided isolation, by puncturing its host plant for an activity test and analyzing on the HPLC-DAD-3D mode for a purity check. Then, the active and pure phytotoxin was characterized as a dirhamnolipid (Rha-Rha-C10-C10) using the NMR, ESIMS, IR and UV methods. The herbicidal activity of dirhamnolipid was evaluated by the inhibition rate on the primary root length and the fresh plant weight of nine test plants, and the synergistic effect when combining with commercial herbicides. Dirhamnolipid exhibited broad herbicidal activity against eight weed species with IC50 values ranging from 28.91 to 217.71 mg L-1 and no toxicity on Oryza sativa, and the herbicidal activity could be synergistically improved combining dirhamnolipid with commercial herbicides. Thus, dirhamnolipid that originated from C. gloeosporioides BWH-1 displayed the potential to be used as a bioherbicide alone, or as an adjuvant added into commercial herbicides, leading to a decrease in herbicides concentration and increased control efficiency.

Participant selection for lung cancer screening by risk modelling (the Pan-Canadian Early Detection of Lung Cancer [PanCan] study): a single-arm, prospective study.

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<0·0001). Compared with 593 (57%) of 1040 lung cancers observed in NLST, 133 (77%) of 172 lung cancers in the PanCan Study were early stage (I or II; p<0·0001). 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.

Predicting EGFR mutation status in lung cancer:Proposal for a scoring model using imaging and demographic characteristics.

OBJECTIVE: To determine if a combination of CT and demographic features can predict EGFR mutation status in bronchogenic carcinoma. METHODS: We reviewed demographic and CT features for patients with molecular profiling for resected non-small cell lung carcinoma. Using multivariate logistic regression, we identified features predictive of EGFR mutation. Prognostic factors identified from the logistic regression model were then used to build a more practical scoring system. RESULTS: A scoring system awarding 5 points for no or minimal smoking history, 3 points for tumours with ground glass component, 3 points for airbronchograms, 2 points for absence of preoperative evidence of nodal enlargement or metastases and 1 point for doubling time of more than a year, resulted in an AUROC of 0.861. A total score of at least 8 yielded a specificity of 95 %. On multivariate analysis sex was not found to be predictor of EGFR status. CONCLUSIONS: A weighted scoring system combining imaging and demographic data holds promise as a predictor of EGFR status. Further studies are necessary to determine reproducibility in other patient groups. A predictive score may help determine which patients would benefit from molecular profiling and may help inform treatment decisions when molecular profiling is not possible. KEY POINTS: • EGFR mutation-targeted chemotherapy for bronchogenic carcinoma has a high success rate. • Mutation testing is not possible in all patients. • EGFR associations include subsolid density, slow tumour growth and minimal/no smoking history. • Demographic or imaging features alone are weak predictors of EGFR status. • A scoring system, using imaging and demographic features, is more predictive.

Cystic fibrosis transmembrane conductance regulator dysfunction in VIP knockout mice.

Vasoactive intestinal peptide (VIP), a neuropeptide, controls multiple functions in exocrine tissues, including inflammation, and relaxation of airway and vascular smooth muscles, and regulates CFTR-dependent secretion, which contributes to mucus hydration and local innate defense of the lung. We had previously reported that VIP stimulates the VPAC1 receptor, PKCϵ signaling cascade, and increases CFTR stability and function at the apical membrane of airway epithelial cells by reducing its internalization rate. Moreover, prolonged VIP stimulation corrects the molecular defects associated with F508del, the most common CFTR mutation responsible for the genetic disease cystic fibrosis. In the present study, we have examined the impact of the absence of VIP on CFTR maturation, cellular localization, and function in vivo using VIP knockout mice. We have conducted pathological assessments and detected signs of lung and intestinal disease. Immunodetection methods have shown that the absence of VIP results in CFTR intracellular retention despite normal expression and maturation levels. A subsequent loss of CFTR-dependent chloride current was measured in functional assays with Ussing chamber analysis of the small intestine ex vivo, creating a cystic fibrosis-like condition. Interestingly, intraperitoneal administration of VIP corrected tissue abnormalities, close to the wild-type phenotype, as well as associated defects in the vital CFTR protein. The results show in vivo a primary role for VIP chronic exposure in CFTR membrane stability and function and confirm in vitro data.

Protective Efficacy of a Novel DNA Vaccine with a CL264 Molecular Adjuvant against Toxoplasma gondii in a Murine Model.

Toxoplasmosis is a significant global zoonosis with devastating impacts, and an effective vaccine against toxoplasmosis for humans has not yet been developed. In this study, we designed and formulated a novel DNA vaccine encoding the inhibitor of STAT1 transcriptional activity (IST) of T. gondii utilizing the eukaryotic expression vector pEGFP-N1 for the first time, with CL264 being a molecular adjuvant. Following intramuscular injection of the vaccine into mice, the levels of antibodies and cytokines were assessed to evaluate the immune response. Additionally, mice were challenged with highly virulent RH-strain tachyzoites of T. gondii, and their survival time was observed. The results show that the levels of IgG in serum, the ratio of IgG2a/IgG1 and the levels of IFN-γ in splenocytes of mice were significantly higher in the pEGFP-TgIST group and the pEGFP-TgIST + CL264 group than in the control group. In addition, the proportion of CD4+/CD8+ T cells was higher in mice immunized with either the pEGFP-TgIST group (p < 0.001) or the pEGFP-TgIST + CL264 group (p < 0.05) compared to the three control groups. Notably, TgIST-immunized mice exhibited prolonged survival times after T. gondii RH strain infection (p < 0.05). Our findings collectively demonstrate that the TgIST DNA vaccine elicits a significant humoral and cellular immune response and offers partial protection against acute T. gondii infection in the immunized mice, which suggests that TgIST holds potential as a candidate for further development as a DNA vaccine.

Screening Criteria Evaluation for Expansion in Pulmonary Neoplasias (SCREEN).

The SCREEN study investigated screening eligibility and survival outcomes between heavy smokers and light-or-never-smokers with lung cancer to determine whether expanded risk factor analysis is needed to refine screening criteria. SCREEN is a retrospective study of 917 lung cancer patients diagnosed between 2005 and 2018 in Nova Scotia, Canada. Screening eligibility was determined using the National Lung Screening Trial (NSLT) criteria. Mortality risk between heavy smokers and light-or-never-smokers was compared using proportional-hazards models. The median follow-up was 2.9 years. The cohort was comprised of 179 (46.1%) female heavy smokers and 306 (57.8%) female light-or-never-smokers. Light-or-never-smokers were more likely to have a diagnosis of adenocarcinoma [n=378 (71.6%)] compared to heavy smokers [n=234 (60.5%); P< 0.001]. Heavy smokers were more frequently diagnosed with squamous cell carcinoma [n=111 (28.7%)] compared to light-or-never-smokers, [n=100 (18.9%); P< 0.001]. Overall, 36.9% (338) of patients met NLST screening criteria. There was no difference in 5-year survival between light-or-never-smokers and heavy smokers [55.2% (338) vs 58.5% (529); P = 0.408; HR 1.06, 95% CI 0.80-1.40; P = 0.704]. Multivariate analysis showed that males had an increased mortality risk [HR 2.00 (95% CI 1.57-2.54); P< 0.001]. Half of lung cancer patients were missed with the conventional screening criteria. There were more curable, stage 1 tumors among light-or-never-smokers. Smoking status and age alone may be insufficient predictors of lung cancer risk and prognosis. Expanded risk factor analysis is needed to refine lung cancer screening criteria.

The role of atypical pathogens in community-acquired pneumonia.

The term atypical pneumonia was first used in 1938, and by the 1970s it was widely used to refer to pneumonia due to Mycoplasma pneumoniae, Legionella pneumophila (or other Legionella species), and Chlamydophila pneumoniae. However, in the purest sense all pneumonias other than the classic bacterial pneumonias are atypical. Currently many favor abolition of the term atypical pneumonia.This review categorizes atypical pneumonia pathogens as conventional ones; viral agents and emerging atypical pneumonia pathogens. We emphasize viral pneumonia because with the increasing availability of multiplex polymerase chain reaction we can identify the agent(s) responsible for viral pneumonia. By using a sensitive assay for procalcitonin one can distinguish between viral and bacterial pneumonia. This allows pneumonia to be categorized as bacterial or viral at the time of admission to hospital or at discharge from the emergency department and soon thereafter further classified as to the etiology, which should be stated as definite or probable.

Lung-Protective Ventilation Attenuates Mechanical Injury While Hypercapnia Attenuates Biological Injury in a Rat Model of Ventilator-Associated Lung Injury.

Background and Objective: Lung-protective mechanical ventilation is known to attenuate ventilator-associated lung injury (VALI), but often at the expense of hypoventilation and hypercapnia. It remains unclear whether the main mechanism by which VALI is attenuated is a product of limiting mechanical forces to the lung during ventilation, or a direct biological effect of hypercapnia. Methods: Acute lung injury (ALI) was induced in 60 anesthetized rats by the instillation of 1.25 M HCl into the lungs via tracheostomy. Ten rats each were randomly assigned to one of six experimental groups and ventilated for 4 h with: 1) Conventional HighV E Normocapnia (high VT, high minute ventilation, normocapnia), 2) Conventional Normocapnia (high VT, normocapnia), 3) Protective Normocapnia (VT 8 ml/kg, high RR), 4) Conventional iCO 2 Hypercapnia (high VT, low RR, inhaled CO2), 5) Protective iCO 2 Hypercapnia (VT 8 ml/kg, high RR, added CO2), 6) Protective endogenous Hypercapnia (VT 8 ml/kg, low RR). Blood gasses, broncho-alveolar lavage fluid (BALF), and tissue specimens were collected and analyzed for histologic and biologic lung injury assessment. Results: Mild ALI was achieved in all groups characterized by a decreased mean PaO2/FiO2 ratio from 428 to 242 mmHg (p < 0.05), and an increased mean elastance from 2.46 to 4.32 cmH2O/L (p < 0.0001). There were no differences in gas exchange among groups. Wet-to-dry ratios and formation of hyaline membranes were significantly lower in low VT groups compared to conventional tidal volumes. Hypercapnia reduced diffuse alveolar damage and IL-6 levels in the BALF, which was also true when CO2 was added to conventional VT. In low VT groups, hypercapnia did not induce any further protective effect except increasing pulmonary IL-10 in the BALF. No differences in lung injury were observed when hypercapnia was induced by adding CO2 or decreasing minute ventilation, although permissive hypercapnia decreased the pH significantly and decreased liver histologic injury. Conclusion: Our findings suggest that low tidal volume ventilation likely attenuates VALI by limiting mechanical damage to the lung, while hypercapnia attenuates VALI by limiting pro-inflammatory and biochemical mechanisms of injury. When combined, both lung-protective ventilation and hypercapnia have the potential to exert an synergistic effect for the prevention of VALI.

Consensus Recommendations to Optimize Testing for New Targetable Alterations in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) has historically been associated with a poor prognosis and low 5-year survival, but the use of targeted therapies in NSCLC has improved patient outcomes over the past 10 years. The pace of development of new targeted therapies is accelerating, with the associated need for molecular testing of new targetable alterations. As the complexity of biomarker testing in NSCLC increases, there is a need for guidance on how to manage the fluid standard-of-care in NSCLC, identify pragmatic molecular testing requirements, and optimize result reporting. An expert multidisciplinary working group with representation from medical oncology, pathology, and clinical genetics convened via virtual meetings to create consensus recommendations for testing of new targetable alterations in NSCLC. The importance of accurate and timely testing of all targetable alterations to optimize disease management using targeted therapies was emphasized by the working group. Therefore, the panel of experts recommends that all targetable alterations be tested reflexively at NSCLC diagnosis as part of a comprehensive panel, using methods that can detect all relevant targetable alterations. In addition, comprehensive biomarker testing should be performed at the request of the treating clinician upon development of resistance to targeted therapy. The expert multidisciplinary working group also made recommendations for reporting to improve clarity and ease of interpretation of results by treating clinicians and to accommodate the rapid evolution in clinical actionability of these alterations. Molecular testing of all targetable alterations in NSCLC is the key for treatment decision-making and access to new therapies. These consensus recommendations are intended as a guide to further optimize molecular testing of new targetable alterations.

Evaluating Targeted Next-Generation Sequencing Assays and Reference Materials for NTRK Fusion Detection.

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from tropomyosin receptor kinase inhibitors is recommended, but the current NTRK testing landscape, including next-generation sequencing (NGS), is fragmented and availability of assays varies widely. The analytical and clinical performance of four commonly available RNA-based NGS assays, Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines and SeraSeq formalin-fixed, paraffin-embedded reference material], NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples, according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control-validated clinical samples was 100% for all assays. This comparison of the strengths and limitations of four RNA-based NGS assays will inform physicians and pathologists regarding optimal assay selection to identify patients with NTRK fusion-positive tumors.

Physiologic and biologic characteristics of three experimental models of acute lung injury in rats.

BACKGROUND: Strategies to attenuate ventilator-associated lung injury have been tested in various experimental methods of acute lung injury (ALI). Conclusions are often drawn from physiologic and biologic effects, but the influence of the model on these results is not known. Our aim in this study was to characterize frequently used models of experimental ALI. METHODS: Twenty Sprague Dawley rats were anesthetized and their lungs mechanically ventilated for 5 hours. Three models of ALI (surfactant washout, acid aspiration, and high tidal volume ventilation) were investigated with regard to hemodynamics, respiratory mechanics, gas exchange, lung pathology, and inflammatory reactions. Animals without ALI served as controls. RESULTS: Five animals in each group were analyzed. Dynamic compliance and Pao(2)/fraction of inspired oxygen ratio decreased by at least 50% in all groups after 1 hour. Whereas compliance remained decreased in all models, oxygenation returned to baseline values in the lavage group after 5 hours. Diffuse alveolar damage was worse in the high tidal volume model and was not different between the control and lavage animals. Interleukin-6 was increased in bronchoalveolar lavage fluid in the aspiration and high tidal volume models. CONCLUSIONS: Although comparable physiologic effects meeting acute respiratory distress syndrome criteria were achieved in all models, the biologic responses varied among lung injury models. The acid aspiration model created both respiratory and inflammatory responses typically seen in ALI; these data suggest that it may be the most clinically applicable model to study the intermediate-term effects of ventilator-associated lung injury in rats.

Single-cell RNA sequencing for the identification of early-stage lung cancer biomarkers from circulating blood.

Lung cancer accounts for more than half of the new cancers diagnosed world-wide with poor survival rates. Despite the development of chemical, radiological, and immunotherapies, many patients do not benefit from these therapies, as recurrence is common. We performed single-cell RNA-sequencing (scRNA-seq) analysis using Fluidigm C1 systems to characterize human lung cancer transcriptomes at single-cell resolution. Validation of scRNA-seq differentially expressed genes (DEGs) through quantitative real time-polymerase chain reaction (qRT-PCR) found a positive correlation in fold-change values between C-X-C motif chemokine ligand 1 (CXCL1) and 2 (CXCL2) compared with bulk-cell level in 34 primary lung adenocarcinomas (LUADs) from Stage I patients. Furthermore, we discovered an inverse correlation between chemokine mRNAs, miR-532-5p, and miR-1266-3p in early-stage primary LUADs. Specially, miR-532-5p was quantifiable in plasma from the corresponding LUADs. Collectively, we identified markers of early-stage lung cancer that were validated in primary lung tumors and circulating blood.

Profiling non-small cell lung cancer reveals that PD-L1 is associated with wild type EGFR and vascular invasion, and immunohistochemistry quantification of PD-L1 correlates weakly with RT-qPCR.

Most lung cancer patients are diagnosed at an advanced stage, limiting their treatment options with very low response rate. Lung cancer is the most common cause of cancer death worldwide. Therapies that target driver gene mutations (e.g. EGFR, ALK, ROS1) and checkpoint inhibitors such anti-PD-1 and PD-L1 immunotherapies are being used to treat lung cancer patients. Identification of correlations between driver mutations and PD-L1 expression will allow for the best management of patient treatment. 851 cases of non-small cell lung cancer cases were profiled for the presence of biomarkers EGFR, KRAS, BRAF, and PIK3CA mutations by SNaPshot/sizing genotyping. Immunohistochemistry was used to identify the protein expression of ALK and PD-L1. Total PD-L1 mRNA expression (from unsorted tumor samples) was quantified by RT-qPCR in a sub-group of the cohort to assess its correlation with PD-L1 protein level in tumor cells. Statistical analysis revealed correlations between the presence of the mutations, PD-L1 expression, and the pathological data. Specifically, increased PD-L1 expression was associated with wildtype EGFR and vascular invasion, and total PD-L1 mRNA levels correlated weakly with protein expression on tumor cells. These data provide insights into driver gene mutations and immune checkpoint status in relation to lung cancer subtypes and suggest that RT-qPCR is useful for assessing PD-L1 levels.

Canadian ROS proto-oncogene 1 study (CROS) for multi-institutional implementation of ROS1 testing in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) harboring ROS proto-oncogene 1 (ROS1) gene rearrangements show dramatic response to the tyrosine kinase inhibitor (TKI) crizotinib. Current best practice guidelines recommend that all advanced stage non-squamous NSCLC patients be also tested for ROS1 gene rearrangements. Several studies have suggested that ROS1 immunohistochemistry (IHC) using the D4D6 antibody may be used to screen for ROS1 fusion positive lung cancers, with assays showing high sensitivity but moderate to high specificity. A break apart fluorescence in situ hybridization (FISH) test is then used to confirm the presence of ROS1 gene rearrangement. The goal of Canadian ROS1 (CROS) study was to harmonize ROS1 laboratory developed testing (LDT) by using IHC and FISH assays to detect ROS1 rearranged lung cancers across Canadian pathology laboratories. Cell lines expressing different levels of ROS1 (high, low, none) were used to calibrate IHC protocols after which participating laboratories ran the calibrated protocols on a reference set of 24 NSCLC cases (9 ROS1 rearranged tumors and 15 ROS1 non-rearranged tumors as determined by FISH). Results were compared using a centralized readout. The stained slides were evaluated for the cellular localization of staining, intensity of staining, the presence of staining in non-tumor cells, the presence of non-specific staining (e.g. necrosis, extracellular mater, other) and the percent positive cells. H-score was also determined for each tumor. Analytical sensitivity and specificity harmonization was achieved by using low limit of detection (LOD) as either any positivity in the U118 cell line or H-score of 200 with the HCC78 cell line. An overall diagnostic sensitivity and specificity of up to 100% and 99% respectively was achieved for ROS1 IHC testing (relative to FISH) using an adjusted H-score readout on the reference cases. This study confirms that LDT ROS1 IHC assays can be highly sensitive and specific for detection of ROS1 rearrangements in NSCLC. As NSCLC can demonstrate ROS1 IHC positivity in FISH-negative cases, the degree of the specificity of the IHC assay, especially in highly sensitive protocols, is mostly dependent on the readout cut-off threshold. As ROS1 IHC is a screening assay for a rare rearrangements in NSCLC, we recommend adjustment of the readout threshold in order to balance specificity, rather than decreasing the overall analytical and diagnostic sensitivity of the protocols.

Regulation of Nrf2/ARE Pathway by Dietary Flavonoids: A Friend or Foe for Cancer Management?

The nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway is an important cell signaling mechanism in maintaining redox homeostasis in humans. The role of dietary flavonoids in activating Nrf2/ARE in relation to cancer chemoprevention or cancer promotion is not well established. Here we summarize the dual effects of flavonoids in cancer chemoprevention and cancer promotion with respect to the regulation of the Nrf2/ARE pathway, while underlying the possible cellular mechanisms. Luteolin, apigenin, quercetin, myricetin, rutin, naringenin, epicatechin, and genistein activate the Nrf2/ARE pathway in both normal and cancer cells. The hormetic effect of flavonoids has been observed due to their antioxidant or prooxidant activity, depending on the concentrations. Reported in vitro and in vivo investigations suggest that the activation of the Nrf2/ARE pathway by either endogenous or exogenous stimuli under normal physiological conditions contributes to redox homeostasis, which may provide a mechanism for cancer chemoprevention. However, some flavonoids, such as luteolin, apigenin, myricetin, quercetin, naringenin, epicatechin, genistein, and daidzein, at low concentrations (1.5 to 20 µM) facilitate cancer cell growth and proliferation in vitro. Paradoxically, some flavonoids, including luteolin, apigenin, and chrysin, inhibit the Nrf2/ARE pathway in vitro. Therefore, even though flavonoids play a major role in cancer chemoprevention, due to their possible inducement of cancer cell growth, the effects of dietary flavonoids on cancer pathophysiology in patients or appropriate experimental animal models should be investigated systematically.