•No technical bias between fresh frozen and FFPE samples.•A high fraction of adenocarcinoma patients with activating KRAS mutations.•Mutations in TP53, STK11 and SMARCA4 linked to poor prognosis in ...adenocarcinoma.•Mutations in CSMD3 linked to better prognosis in squamous cell carcinoma.•Co-mutations in TP53 or STK11 confer poor prognosis in KRAS positive patients.
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.
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.
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.
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.
Tumor‐associated macrophages (TAMs) are attractive targets for immunotherapy. Recently, studies in animal models showed that treatment with an anti‐TAM antibody directed against the scavenger ...receptor MARCO resulted in suppression of tumor growth and metastatic dissemination. Here we investigated the expression of MARCO in relation to other macrophage markers and immune pathways in a non‐small cell lung cancer (NSCLC) cohort (n = 352). MARCO, CD68, CD163, MSR1 and programmed death ligand‐1 (PD‐L1) were analyzed by immunohistochemistry and immunofluorescence, and associations to other immune cells and regulatory pathways were studied in a subset of cases (n = 199) with available RNA‐seq data. We observed a large variation in macrophage density between cases and a strong correlation between CD68 and CD163, suggesting that the majority of TAMs present in NSCLC exhibit a protumor phenotype. Correlation to clinical data only showed a weak trend toward worse survival for patients with high macrophage infiltration. Interestingly, MARCO was expressed on a distinct subpopulation of TAMs, which tended to aggregate in close proximity to tumor cell nests. On the transcriptomic level, we found a positive association between MARCO gene expression and general immune response pathways including strong links to immunosuppressive TAMs, T‐cell infiltration and immune checkpoint molecules. Indeed, a higher macrophage infiltration was seen in tumors expressing PD‐L1, and macrophages residing within tumor cell nests co‐expressed MARCO and PD‐L1. Thus, MARCO is a potential new immune target for anti‐TAM treatment in a subset of NSCLC patients, possibly in combination with available immune checkpoint inhibitors.
What's new?
Because of their dual anti‐ and proinflammatory role, tumor‐associated macrophages (TAMs) are attractive targets for immunotherapy. Treatment with antibodies targeting the scavenger receptor MARCO has previously been shown to result in a more proinflammatory macrophage phenotype and reduced tumor growth. Here, the authors show that MARCO defines a distinct subset of immune‐suppressive TAMs in a large lung cancer cohort. A higher macrophage infiltration was observed in tumors expressing PD‐L1, and transcriptomic data revealed a correlation between MARCO, checkpoint molecules and T‐cell infiltration. MARCO could thus be a potential new immune target for anti‐TAM treatment in a subset of NSCLC patients.
•Pleural effusions provide an abundant source for liquid biopsy RNA-profiling.•Exosomal derived microRNAs provide superior diagnostic value compared to mRNAs.•Specific exosomal derived RNAs define ...patients with lung adenocarcinoma.
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.
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.
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).
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.
IntroductionAtrial 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.MethodsStroke 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 disseminationThe 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.
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.
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.
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.
What's new?
New tools are needed in order to improve risk stratification and therapy selection in early‐stage lung adenocarcinoma. Inherent differences in gene expression between adenocarcinoma subtypes could facilitate the development of such tools. The authors of this study derived platform‐independent, single‐sample predictors (SSP) of adenocarcinoma subtypes, based on gene expression. Derived SSPs successfully provided prognostic information in surgically treated stage I lung adenocarcinoma patients. The single‐sample classifier was readily translated into assays applicable to archival tissue, indicating clinical utility. The findings highlight the clinical relevance of transcriptional signatures and gene expression predictors in lung adenocarcinoma, warranting their further investigation and development.
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
), the validated variants rs10491684 in DOCK8 (P-value = 1.95 × 10
), rs6118 in SERPINA5 (P-value = 5.83 × 10
), and rs5877 in SERPINC1 (P-value = 1.07 × 10
), and the genes CAPZA2 (P-value = 4.03 × 10
) and SERPINC1 (P-value = 1.55 × 10
). The SNVs in the top-scoring pathway "Factors involved in megakaryocyte development and platelet production" (P-value = 3.34 × 10
) 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
), and decrease (OR = 66.82, P-value = 5.92 × 10
). 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.
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.
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).
Longitudinal cohort study.
Three secondary-level hospitals.
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.
SLP assessment, advice, and therapy for dysphagia.
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.
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).
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).
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