Refined Diagnosis of Pleural Effusions by Immunocytochemistry of Cell Blocks.

BACKGROUND/AIM: The main objective of microscopic examination of pleural effusions is to ascertain the presence of malignant cells. Effusions prepared routinely using May-Grünwald-Giemsa (MGG)- and Papanicolaou (PAP)-staining can, in a number of cases, provide inconclusive cytological results regarding malignancy. PATIENTS AND METHODS: This report describes the refined diagnosis of such cases based on immunocytochemical analysis of pleural effusion cell blocks. Of the 340 pleural effusions obtained during 2019 at the Department of Clinical Cytology, Gävle Hospital, Sweden, 63 (18.5%) contained atypical cells of undetermined significance or potentially malignant cells. RESULTS: This diagnosis could be refined using Epithelial Cell Adhesion Molecule/EPCAM (BEREP4) immunocytochemical analysis of effusion cell blocks, allowing previously inconclusive effusions to be classified as clearly benign 42/63 (66.7%) or malignant 21/63 (33.3%). Effusions initially diagnosed as clearly malignant (27/340; 7.9%) were all 27 (100%) BEREP4-immuno-stained. Most BEREP4-positive effusions (37/48; 77.1%) were also carcinoembryonic antigen (CEA) positive. The number of BEREP4-positive cells, however, tended to exceed that of CEA-positive cells. The BEREP4 positive effusions were further examined using different monoclonal antibodies, such as Thyroid transcription factor 1 (TTF-1) for primary pulmonary adenocarcinoma, to determine the original site of the primary tumour. CONCLUSION: Immunohistochemical staining of pleural effusion cell blocks significantly refines the diagnosis of serous pleural effusions, especially in cases where the preliminary diagnosis was atypical cells of undetermined significance or potentially malignant cells. Furthermore, in the cases of malignancy, the origin of the primary tumour could most often be determined.

Highly elevated systemic inflammation is a strong independent predictor of early mortality in advanced non-small cell lung cancer.

BACKGROUND: Ample evidence support inflammation as a marker of outcome in non-small cell lung cancer (NSCLC). Here we explore the outcome for a subgroup of patients with advanced disease and substantially elevated systemic inflammatory activity. METHODS: The source cohort included consecutive patients diagnosed with NSCLC between January 2016 - May 2017 (n = 155). Patients with active infection were excluded. Blood parameters were examined individually, and cut-offs (ESR > 60 mm, CRP > 20 mg/L, WBC > 10 × 109, PLT > 400 × 109) were set to define the group of hyperinflamed patients. A score was developed by assigning one point for each parameter above cut-off (0-4 points). RESULTS: High systemic inflammation was associated with advanced stage and was seldom present in limited NSCLC. However, the one year survival of patients in stage IIIB-IV (n = 93) with an inflammation score of ≥2 was 0% compared to 33% and 50% among patients with a score of 1 and 0 respectively. The effect of a high inflammation score on overall survival remained significant in multi-variate analysis adjusted for confounding factors. The independent hazard ratio of an inflammation score ≥ 2 in multi-variate analysis (HR 3.43, CI 1.76-6.71) was comparable to a change in ECOG PS from 0 to 2 (HR 2.42, CI 1.13-5.18). CONCLUSION: Our results show that high level systemic inflammation is a strong independent predictor of poor survival in advanced stage NSCLC. This observation may indicate a need to use hyperinflammation as an additional clinical parameter for stratification of patients in clinical studies and warrants further research on underlying mechanisms linked to tumor progression.

The association of four genetic variants with myelosuppression in gemcitabine-treated Japanese is not evident in gemcitabine/carboplatin-treated Swedes.

Gemcitabine/carboplatin-induced myelosuppressive adverse drug reactions (ADRs) are clinical problems leading to patient suffering and dose alterations. There is a need for personalised medicine to improve treatment effects and patients' well-being. We tested four genetic variants, rs11141915, rs1901440, rs12046844 and rs11719165, previously suggested as potential biomarkers for gemcitabine-induced leukopenia/neutropenia in Japanese patients, in 213 Swedish gemcitabine/carboplatin-treated non-small cell lung cancer (NSCLC) patients. DNA was genotyped using TaqMan probes and real-time PCR. The relationships between the risk alleles and low toxicity (non-ADR: Common Terminology Criteria for Adverse Events [CTCAE] grades 0) or high toxicity (ADR: CTCAE grades 3-4) of platelets, leukocytes and neutrophils were evaluated using Fisher's exact test. The risk alleles did not correlate with myelosuppression, and the strongest borderline significance (not withstanding adjustment for multiple testing) was for rs1901440 (neutropenia, p = 0.043) and rs11719165 (leukopenia, p = 0.049) where the risk alleles trended towards lower toxicity, contrasting with previous study findings. Risk alleles and higher risk scores were more common among our patients. We conclude that the genetic variants do not apply to Swedish patients treated with gemcitabine/carboplatin. However, they can still be important in other populations and cohorts, especially in a gemcitabine monotherapy setting, where the causal genetic variation might influence myelosuppressive ADRs.

Swallowing Function in COVID-19 Patients After Invasive Mechanical Ventilation.

OBJECTIVE: To explore swallowing function and risk factors associated with delayed recovery of swallowing in patients with COVID-19 post-invasive mechanical ventilation using the Functional Oral Intake Scale (FOIS). DESIGN: Longitudinal cohort study. SETTING: Three secondary-level hospitals. PARTICIPANTS: Invasively ventilated patients (N=28) who were hospitalized with severe COVID-19 and referred to the hospitals' speech and language pathology (SLP) departments after mechanical ventilation between March 5 and July 5, 2020 for an evaluation of swallowing function before commencing oral diet. INTERVENTIONS: SLP assessment, advice, and therapy for dysphagia. MAIN OUTCOME MEASURES: Oral intake levels at baseline and hospital discharge according to the FOIS. Patients were stratified according to FOIS (1-5, dysphagia; 6-7, functional oral intake). Data regarding comorbidities, frailty, intubation and tracheostomy, proning, and SLP evaluation were collected. RESULTS: Dysphagia was found in 71% of the patients at baseline (79% men; age, 61±12y; body mass index, 30±8 kg/m2). The median FOIS score at baseline was 2 (interquartile range [IQR], 1) vs 5 (IQR, 2.5) at hospital discharge. Patients with dysphagia were older (64±8.5y vs 53±16y; P=.019), had a higher incidence of hypertension (70% vs 12%; P=.006), and were ventilated invasively longer (16±7d vs 10±2d; P=.017) or had a tracheostomy (9±9d vs 1±2d; P=.03) longer. A negative association was found between swallowing dysfunction at bedside and days hospitalized (r=-0.471, P=.01), and number of days in the intensive care unit (ICU) (r=-0.48, P=.01). CONCLUSION: Dysphagia is prevalent in COVID-19 patients after invasive mechanical ventilation and is associated with number of days in hospital and number of days in the ICU. Swallowing function and tolerance of oral diet improved at discharge (P<.001).

Mesothelioma in immigrants from Turkey: Genes have a minor role.

OBJECTIVE: To examine the incidence and epidemiology of malignant mesothelioma in immigrants from Karain where there is an extraordinarily high incidence of mesothelioma, Cappadocia, Turkey, to Stockholm, Sweden, and their children over 20 years of age born in Stockholm, i.e. two genetically similar populations with and without erionite exposure. METHODS: This survey was conducted as a retrospective cohort study. Standardized average annual mesothelioma incidence rates (AAMIRs) and mesothelioma standardized incidence ratio (mSIR) were calculated. Cox regression analysis was used to determine the importance of different factors related to mesothelioma risk. RESULTS: The cohort consisted of 337 people, 203 of whom were born and/or lived in Karain before immigrating to Sweden (erionite-exposed), and 134 who were born in Stockholm (erionite-unexposed). There were 69 deaths, 42 (61%) due to mesothelioma, and two patients with the disease who were still alive. Of these 44 patients, 22 were men. All mesothelioma patients were in the erionite-exposed group. In the age group 30-49 years, mesothelioma developed in 11 of 38 (29%) with erionite exposure, while there were no cases among 86 persons in the non-exposed group. For men, the AAMIR was 253.9 per 100,000 persons in the whole cohort, and for women, it was 350.9. The mSIR was 71.9 for men and 393.1 for women. Exposure to erionite exceeding 20 years and age over 40 years were associated with increased mesothelioma risk. CONCLUSION: Exposure to erionite is the leading cause of mesothelioma in Karain villagers, and genetic factors are probably of minor importance.

Determination and Pharmacokinetics of Omeprazole Enantiomers in Human Plasma and Oral Fluid Utilizing Microextraction by Packed Sorbent and Liquid Chromatography-Tandem Mass Spectrometry.

In the present work, the determination of omeprazole (OME) enantiomers in oral fluid and plasma samples was carried out utilizing microextraction by packed sorbent (MEPS) and liquid chromatography-tandem mass spectrometry. A chiral column with cellulose-SB phase was used for the first time for enantiomeric separation of OME with an isocratic elution system using 0.2% ammonium hydroxide in hexane-ethanol mixture (70 : 30, v/v) as the mobile phase. OME enantiomers were determined utilizing a triple quadrupole tandem mass spectrometer in positive ion mode (ESI+) monitoring mass transitions: m/z 346.3 ⟶ 198.0 for OME and m/z 369.98 ⟶ 252.0 for internal standard. The limits of detection and quantification of the present method for both enantiomers were 0.1 and 0.4 ng/mL, respectively. The method validation provided good accuracy and precision. The matrix effect factor was less than 5%, and no interfering peaks were observed. The interday precision values ranged from 2.2 to 7.5 (%RSD), and the accuracy of determinations varied from -9.9% to 8.3%. In addition, the pharmacokinetics (PK) of omeprazole enantiomers in healthy subjects after a single oral dose was investigated. (S)-Enantiomers showed higher levels than (R)-enantiomers throughout 24 h. It was found that the mean maximum concentrations of (R)- and (S)-omeprazole in plasma samples were about two times higher than in oral fluid.

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

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

Whole-genome sequencing and gene network modules predict gemcitabine/carboplatin-induced myelosuppression in non-small cell lung cancer patients.

Gemcitabine/carboplatin chemotherapy commonly induces myelosuppression, including neutropenia, leukopenia, and thrombocytopenia. Predicting patients at risk of these adverse drug reactions (ADRs) and adjusting treatments accordingly is a long-term goal of personalized medicine. This study used whole-genome sequencing (WGS) of blood samples from 96 gemcitabine/carboplatin-treated non-small cell lung cancer (NSCLC) patients and gene network modules for predicting myelosuppression. Association of genetic variants in PLINK found 4594, 5019, and 5066 autosomal SNVs/INDELs with p ≤ 1 × 10-3 for neutropenia, leukopenia, and thrombocytopenia, respectively. Based on the SNVs/INDELs we identified the toxicity module, consisting of 215 unique overlapping genes inferred from MCODE-generated gene network modules of 350, 345, and 313 genes, respectively. These module genes showed enrichment for differentially expressed genes in rat bone marrow, human bone marrow, and human cell lines exposed to carboplatin and gemcitabine (p < 0.05). Then using 80% of the patients as training data, random LASSO reduced the number of SNVs/INDELs in the toxicity module into a feasible prediction model consisting of 62 SNVs/INDELs that accurately predict both the training and the test (remaining 20%) data with high (CTCAE 3-4) and low (CTCAE 0-1) maximal myelosuppressive toxicity completely, with the receiver-operating characteristic (ROC) area under the curve (AUC) of 100%. The present study shows how WGS, gene network modules, and random LASSO can be used to develop a feasible and tested model for predicting myelosuppressive toxicity. Although the proposed model predicts myelosuppression in this study, further evaluation in other studies is required to determine its reproducibility, usability, and clinical effect.

Intrabronchial stents in heterogenous emphysema: a highly selected material followed 5 years.

BACKGROUND: Endobronchial lung volume reduction (EBVR) with one-way valves introduced into the most diseased lobe of the lung is a non-invasive method to improve lung function in patients with severe heterogenous emphysema. The problem is to select the right patients for the procedure. Furthermore, the long-term effects have not been reported in most studies. METHODS: EBVR was performed in 35 patients with severely handicapping emphysema and with one radiologically clearly enlarged (at least 125%) target lung lobe and clearly visible and complete interlobar fissures on CT. Most of the successful survivors have then been followed for five years. RESULTS: Five (14%) were primary failures (defined as less than 15% increase of FEV1sec) and 10 (28%) were secondary failures (the valves were coughed up or removed for some reason). In the 21 successful l patients, the improvement of FEV1sec was in the mean 59%. Over the years, FEV1sec gradually decreased but was still higher 5 years later than initially. Of the successful group, 76% were still alive after 5 years, while only 50% were in the refused or failed groups. CONCLUSIONS: With careful selection of patients remarkably good results in lung function can be achieved, and these improvements will last for years though slowly decrease. In addition, the results indicate an improvement in survival with successful EBVR procedure.

Genetic association of gemcitabine/carboplatin-induced leukopenia and neutropenia in non-small cell lung cancer patients using whole-exome sequencing.

OBJECTIVES: Gemcitabine/carboplatin treatment is known to cause severe adverse drug reactions which can lead to the need for reduction or cessation of chemotherapy. It would be beneficial to identify patients at risk of severe hematological toxicity in advance before treatment start. This study aims to identify genetic markers for gemcitabine/carboplatin-induced leukopenia and neutropenia in non-small cell lung cancer patients. MATERIAL AND METHODS: Whole-exome sequencing was performed on 215 patients. Association analysis was performed on single-nucleotide variants (SNVs) and genes, and the validation was based on an independent genome-wide association study (GWAS). Based on the association and validation analyses the genetic variants were then selected for and used in weighted genetic risk score (wGRS) prediction models for leukopenia and neutropenia. RESULTS: Association analysis identified 50 and 111 SNVs, and 12 and 20 genes, for leukopenia and neutropenia, respectively. Of these SNVS 20 and 19 were partially validated for leukopenia and neutropenia, respectively. The genes SVIL (p = 2.48E-06) and EFCAB2 (p = 4.63E-06) were significantly associated with leukopenia contain the partially validated SNVs rs3740003, rs10160013, rs1547169, rs10927386 and rs10927387. The wGRS prediction models showed significantly different risk scores for high and low toxicity patients. CONCLUSION: We have identified and partially validated genetic biomarkers in SNVs and genes correlated to gemcitabine/carboplatin-induced leukopenia and neutropenia and created wGRS models for predicting the risk of chemotherapy-induced hematological toxicity. These results provide a strong foundation for further studies of chemotherapy-induced toxicity.

Genes and variants in hematopoiesis-related pathways are associated with gemcitabine/carboplatin-induced thrombocytopenia.

Chemotherapy-induced myelosuppression, including thrombocytopenia, is a recurrent problem during cancer treatments that may require dose alterations or cessations that could affect the antitumor effect of the treatment. To identify genetic markers associated with treatment-induced thrombocytopenia, we whole-exome sequenced 215 non-small cell lung cancer patients homogeneously treated with gemcitabine/carboplatin. The decrease in platelets (defined as nadir/baseline) was used to assess treatment-induced thrombocytopenia. Association between germline genetic variants and thrombocytopenia was analyzed at single-nucleotide variant (SNV) (based on the optimal false discovery rate, the severity of predicted consequence, and effect), gene, and pathway levels. These analyses identified 130 SNVs/INDELs and 25 genes associated with thrombocytopenia (P-value < 0.002). Twenty-three SNVs were validated in an independent genome-wide association study (GWAS). The top associations include rs34491125 in JMJD1C (P-value = 9.07 × 10-5), the validated variants rs10491684 in DOCK8 (P-value = 1.95 × 10-4), rs6118 in SERPINA5 (P-value = 5.83 × 10-4), and rs5877 in SERPINC1 (P-value = 1.07 × 10-3), and the genes CAPZA2 (P-value = 4.03 × 10-4) and SERPINC1 (P-value = 1.55 × 10-3). The SNVs in the top-scoring pathway "Factors involved in megakaryocyte development and platelet production" (P-value = 3.34 × 10-4) were used to construct weighted genetic risk score (wGRS) and logistic regression models that predict thrombocytopenia. The wGRS predict which patients are at high or low toxicity risk levels, for CTCAE (odds ratio (OR) = 22.35, P-value = 1.55 × 10-8), and decrease (OR = 66.82, P-value = 5.92 × 10-9). The logistic regression models predict CTCAE grades 3-4 (receiver operator characteristics (ROC) area under the curve (AUC) = 0.79), and large decrease (ROC AUC = 0.86). We identified and validated genetic variations within hematopoiesis-related pathways that provide a solid foundation for future studies using genetic markers for predicting chemotherapy-induced thrombocytopenia and personalizing treatments.

Subjective swallowing symptoms and related risk factors in COPD.

OBJECTIVES: This study aimed to investigate the prevalence of subjective (i.e. self-reported) swallowing symptoms in a large cohort of patients with stable chronic obstructive pulmonary disease (COPD) and to identify potential related risk factors. METHODS: A total of 571 patients with COPD, investigated in a stable phase, participated in this multicentre study (335 females, 236 males; mean age: 68.6 years (sd 7.7)). Data were derived from spirometry, a questionnaire and a 30-metre walking test. RESULTS: In total, 33% (n=186) patients reported at least some degree of swallowing problem. The most frequently reported symptom was food lodging in the throat (23%). A significant relationship was found between swallowing symptoms and dyspnoea, assessed as modified Medical Research Council (mMRC) ≥2 compared with <2 (46% versus 22%; p<0.001) and health-related quality of life, assessed as the COPD Assessment Test (CAT) ≥10 (40% versus 19%; p<0.001). Swallowing problems were also related to lower physical capacity (p=0.02) but not to lung function (p>0.28). CONCLUSION: Subjective swallowing symptoms seem to be a common problem in patients with stable COPD. This problem is seen in all stages of the disease, but is more common in symptomatic patients and in patients with lower physical capacity.

Erlotinib treatment induces cytochrome P450 3A activity in non-small cell lung cancer patients.

AIMS: Erlotinib is a tyrosine kinase inhibitor used in the treatment of non-small cell lung cancer highly metabolized by the cytochrome P450 (CYP) 3A. Hence, CYP3A4 activity might be a useful predictor of erlotinib pharmacokinetics in personalized medicine. The effect of erlotinib on CYP3A activity was therefore studied in non-small cell lung cancer patients. METHODS: The study included 32 patients scheduled for erlotinib monotherapy. CYP3A activity was assessed using quinine as a probe before and during erlotinib treatment. Plasma from blood samples drawn 16 hours post quinine administration were analysed using HPLC with fluorescence detection to determine the quinine/3-OH-quinine ratio. RESULTS: Matched samples, available from 13 patients, showed an induction of CYP3A activity (P = 0.003, Wilcoxon's signed rank test) after 2 months of treatment. The quinine/3-OH-quinine ratio decreased from 20.2 (± 13.4) at baseline to 11.0 (± 4.34). Single-point samples, available from 19 patients, supported the decrease in ratio (P = 0.007, Mann-Whitney U-test). Generally, females had a higher CYP3A activity both at baseline and after two months of treatment. Statistical analysis by gender also showed significant increase in CYP3A activity (males, n = 10, P = 0.001, and females, n = 22, P = 0.001). CONCLUSIONS: An induction of CYP3A activity was observed after 2 months of erlotinib treatment which was also seen when subdividing based on gender. It could be important to take this into consideration for patients co-administering other CYP3A-metabolizing drugs during erlotinib treatment and also makes it difficult to use baseline CYP3A activity to predict erlotinib pharmacokinetics.

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

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

Mutation patterns in a population-based non-small cell lung cancer cohort and prognostic impact of concomitant mutations in KRAS and TP53 or STK11.

OBJECTIVES: Non-small cell lung cancer (NSCLC) is a heterogeneous disease with unique combinations of somatic molecular alterations in individual patients, as well as significant differences in populations across the world with regard to mutation spectra and mutation frequencies. Here we aim to describe mutational patterns and linked clinical parameters in a population-based NSCLC cohort. MATERIALS AND METHODS: Using targeted resequencing the mutational status of 82 genes was evaluated in a consecutive Swedish surgical NSCLC cohort, consisting of 352 patient samples from either fresh frozen or formalin fixed paraffin embedded (FFPE) tissues. The panel covers all exons of the 82 genes and utilizes reduced target fragment length and two-strand capture making it compatible with degraded FFPE samples. RESULTS: We obtained a uniform sequencing coverage and mutation load across the fresh frozen and FFPE samples by adaption of sequencing depth and bioinformatic pipeline, thereby avoiding a technical bias between these two sample types. At large, the mutation frequencies resembled the frequencies seen in other western populations, except for a high frequency of KRAS hotspot mutations (43%) in adenocarcinoma patients. Worse overall survival was observed for adenocarcinoma patients with a mutation in either TP53, STK11 or SMARCA4. In the adenocarcinoma KRAS-mutated group poor survival appeared to be linked to concomitant TP53 or STK11 mutations, and not to KRAS mutation as a single aberration. Similar results were seen in the analysis of publicly available data from the cBioPortal. In squamous cell carcinoma a worse prognosis could be observed for patients with MLL2 mutations, while CSMD3 mutations were linked to a better prognosis. CONCLUSION: Here we have evaluated the mutational status of a NSCLC cohort. We could not confirm any survival impact of isolated driver mutations. Instead, concurrent mutations in TP53 and STK11 were shown to confer poor survival in the KRAS-positive adenocarcinoma subgroup.

Prognostic Impact of Tumor Cell Programmed Death Ligand 1 Expression and Immune Cell Infiltration in NSCLC.

INTRODUCTION: Infiltration of T and B/plasma cells has been linked to NSCLC prognosis, but this has not been thoroughly investigated in relation to the expression of programmed death ligand 1 (PD-L1). Here, we determine the association of lymphocytes and PD-L1 with overall survival (OS) in two retrospective cohorts of operated NSCLC patients who were not treated with checkpoint inhibitors targeting the programmed death 1/PD-L1 axis. Moreover, we evaluate how PD-L1 positivity and clinicopathologic factors affect the prognostic association of lymphocytes. METHODS: Cluster of differentiation (CD) 3 (CD3)-, CD8-, CD4-, forkhead box P3 (FOXP3)-, CD20-, CD79A-, and immunoglobulin kappa constant (IGKC)-positive immune cells, and tumor PD-L1 positivity, were determined by immunohistochemistry on tissue microarrays (n = 705). Affymetrix data was analyzed for a patient subset, and supplemented with publicly available transcriptomics data (N = 1724). Associations with OS were assessed by Kaplan-Meier plots and uni- and multivariate Cox regression. RESULTS: Higher levels of T and B plasma cells were associated with longer OS (p = 0.004 and p < 0.001, for CD8 and IGKC, respectively). Highly proliferative tumors with few lymphocytes had the worst outcome. No association of PD-L1 positivity with OS was observed in a nonstratified patient population; however, a significant association with shorter OS was observed in never-smokers (p = 0.009 and p = 0.002, 5% and 50% cutoff). Lymphocyte infiltration was not associated with OS in PD-L1-positive tumors (50% cutoff). The prognostic association of lymphocyte infiltration also depended on the patients' smoking history and histologic subtype. CONCLUSIONS: Proliferation, PD-L1 status, smoking history, and histology should be considered if lymphocyte infiltration is to be used as a prognostic biomarker.

Exosomal RNA-profiling of pleural effusions identifies adenocarcinoma patients through elevated miR-200 and LCN2 expression.

HYPOTHESIS: The inherent challenges associated with tissue biopsies from lung have spurred an interest in the use of liquid biopsies. Pleural effusions are one source of liquid biopsy. Recently, extracellular vesicles of endocytic origin, exosomes, have attracted interest as liquid biopsy of tumors as they are thought to be a mirror of their tumor of origin. Here, we aimed to analyze if RNA profiling of exosomes isolated from pleural effusions could differentiate patients with lung adenocarcinoma from patients with benign inflammatory processes. METHODS: Exosomes were isolated from 36 pleural effusions from patients with adenocarcinoma (n = 18) and patients with benign inflammatory processes (n = 18). The two groups were balanced with respect to age and smoking history but with a gender bias towards males in the benign group. Profiling was conducted using RT-qPCR arrays covering 754 microRNAs and 624 mRNAs followed by statistical ranking of differentially regulated transcripts between the two patient cohorts. RESULTS: RNA profiling revealed differential expression of 17 microRNAs and 71 mRNAs in pleural effusions collected from patients with lung adenocarcinoma compared to pleural effusions from benign lung disease. Overall, top differentially expressed microRNAs, including miR-200 family microRNAs, provided a stronger diagnostic power compared to top differentially expressed mRNAs. However, the mRNA transcript encoding Lipocalin-2 (LCN2) displayed the strongest diagnostic power of all analyzed transcripts (AUC: 0.9916). CONCLUSIONS: Our study demonstrates that exosomal RNA profiling from pleural effusions can be used to identify patients with lung adenocarcinoma from individuals with benign processes and further proposes miR-200 microRNAs and LCN2 as diagnostic markers in lung cancer liquid biopsies.

Co-localisation of Glandular and Squamous Cell Markers in Non-small Cell Lung Cancer.

AIM: Accurate classification of lung carcinomas is crucial for selecting appropriate and adequate chemotherapy treatment. In this study, glandular (adenocarcinoma), and squamous cell differentiation were examined in non-small cell lung carcinoma (NSCLC) without obvious light-microscopic signs of squamous and glandular differentiation. MATERIALS AND METHODS: All lung tumours diagnosed as NSCLC (n=61), without obvious squamous or glandular features, were obtained by bronchial biopsy or core biopsy supported by computed tomography. They were diagnosed during 1996-2009, at the Department of Pathology, Gävle Hospital, Sweden. The tumours were examined immunohistochemically with antibodies against CK5/6, p63 (squamous cell markers) and carcinoembryonic antigen (CEA) (adenocarcinoma cell marker). Double immunostaining (p63/CEA) was also performed on individual tumours. RESULTS: The tumours originated from 36 males and 25 females, aged 54-83 years. Pure squamous cell differentiation (CK5/6 positive only) occurred in 34.4% (n=21) tumours and pure adenocarcinoma cell differentiation (CEA positive only) was present in 14.9% (n=9). Tumours with both squamous and adenocarcinoma features (CK5/6 and CEA positive) were most prevalent (47.5%, n=29). Two tumours (3.3%) were negative with both stains (and also synaptophysin negative). Double immunostaining (p63/CEA) revealed that squamous and adenocarcinoma markers were co-localised in cells in certain tumours. CONCLUSION: Co-localisation of squamous and adenocarcinoma markers in the same tumour cell suggests that additional analyses for novel biomarkers of specific lung cancer types may subsequently lead to a refined treatment choice for patients with the goal of improving clinical outcomes.

A protocol for a prospective observational study using chest and thumb ECG: transient ECG assessment in stroke evaluation (TEASE) in Sweden.

INTRODUCTION: Atrial fibrillation (AF) causes ischaemic stroke and based on risk factor evaluation warrants anticoagulation therapy. In stroke survivors, AF is typically detected with short-term ECG monitoring in the stroke unit. Prolonged continuous ECG monitoring requires substantial resources while insertable cardiac monitors are invasive and costly. Chest and thumb ECG could provide an alternative for AF detection poststroke.The primary objective of our study is to assess the incidence of newly diagnosed AF during 28 days of chest and thumb ECG monitoring in cryptogenic stroke. Secondary objectives are to assess health-related quality of life (HRQoL) using short-form health survey (SF-36) and the feasibility of the Coala Heart Monitor in patients who had a stroke. METHODS: Stroke survivors in Region Gävleborg, Sweden, will be eligible for the study from October 2017. Patients with a history of ischaemic stroke without documented AF before or during ECG evaluation in the stroke unit will be evaluated by the chest and thumb ECG system Coala Heart Monitor. The monitoring system is connected to a smartphone application which allows for remote monitoring and prompt advice on clinical management. Over a period of 28 days, patients will be monitored two times a day and may activate the ECG recording at symptoms. On completion, the system is returned by mail. This system offers a possibility to evaluate the presence of AF poststroke, but the feasibility of this system in patients who recently suffered from a stroke is unknown. In addition, HRQoL using SF-36 in comparison to Swedish population norms will be assessed. The feasibility of the Coala Heart Monitor will be assessed by a self-developed questionnaire. ETHICS AND DISSEMINATION: The study was approved by The Regional Ethical Committee in Uppsala (2017/321). The database will be closed after the last follow-up, followed by statistical analyses, interpretation of results and dissemination to a scientific journal. TRIAL REGISTRATION NUMBER: NCT03301662; Pre-results.