DataSheet_1_Case report: BRAF A598-T599insV mutation as a potential resistance mechanism to alectinib in ALK-rearranged lung adenocarcinoma.docx

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) have improved the prognosis of advanced-stage non-small cell lung cancer (NSCLC) with ALK rearrangement, but resistance mechanisms limit their efficacy. We describe the case of a 63-year-old man with a stage cIVA ALK-rearranged lung adenocarcinoma who developed a BRAF A598-T599insV mutation as a potential resistance mechanism to alectinib, a second-generation ALK TKI. He was treated with an association of BRAF and MEK inhibitors but death occurred two months after treatment initiation in a context of tumor progression and toxicity. Based on this first report of BRAF A598-T599insV mutation occurring in lung cancer, we discuss resistance mechanisms to ALK TKIs, implications of BRAF mutation in NSCLC, and BRAF A598-T599insV mutation in other cancers.</p

Additional file 3: of RNA-sequencing in non-small cell lung cancer shows gene downregulation of therapeutic targets in tumor tissue compared to non-malignant lung tissue

Figure S1. Differential methylated sites. Representation of the amount of methylated sites per gene and per patient subset. The amount of hypermethylated sites per gene can be found on the y-axis, both for hypermethylated sites in non-malignant lung tissue (blue) and hypermethylated sites in tumor tissue (red). Associations with histone markers and CpG islands are color coded per methylation site. P-values can be equal across different subsets and genes for non-significant results because of the statistical method used. (XLSM 509 kb

Additional file 1: of RNA-sequencing in non-small cell lung cancer shows gene downregulation of therapeutic targets in tumor tissue compared to non-malignant lung tissue

Table S1. RNA-sequencing results for tumor tissue (a) or non-malignant lung tissue (b). Differential expression of particular genes (top row) between different patient subsets (left column) is represented by p-values and color coded. Blue and green colors indicate more expression of a particular gene for the first patient subset (red) compared to the second subset (yellow and red). (DOCX 21 kb

Lung function decline in COPACETIC according to rs1051730 and rs8034191 genotypes.

#<p>significant compared to the GG genotype for rs1051730 (p = 0.026).</p>*<p>significant compared to AA genotype for rs8034191 (p = 0.009 and p = 0.017, respectively for FEV₁/FVC and MEF₅₀ decline).</p

Characteristics for COPACATIC and LEUVEN at baseline and after three-year follow-up for COPACETIC.

<p>FEV₁: forced expiratory volume in one second; FVC: forced vital capacity; COPD: chronic obstructive pulmonary disease; GOLD: global initiative for chronic obstructive lung disease; N/A: non-applicable. Percentages are column percentages. Of the LEUVEN participants, 653 subjects were diagnosed with COPD (FEV₁/FVC<0.70).</p

Baseline Characteristics for LEUVEN participants stratified for the clinical impact of airflow obstruction.

<p>Abbreviations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053219#pone-0053219-t001" target="_blank">Table 1</a>. Percentages are column percentages. The 366 asymptomatic smokers are population-based participants of the Dutch-Belgian lung cancer screening trial (NELSON). None of them were previously diagnosed with COPD. However, 148 subjects (40.4%) were found to have an obstructive lung function (based on FEV₁/FVC<0.70) at inclusion. Eleven subjects (2%), that were followed-up at the outpatient clinic because of symptoms compatible with COPD, did not fulfill the criterion of COPD (FEV₁/FVC>0.70). Five patients with end-stage COPD were not actively listed for lung transplantation because of their current smoking status at inclusion.</p

Baseline characteristics for the COPACETIC cohort according to rs1051730 and rs8034191 genotypes.

<p>FEV₁: forced expiratory volume in one second; FVC: forced vital capacity; MEF₅₀ maximum expiratory flow when 50% of the FVC has been exhaled; GOLD: global initiative for chronic obstructive lung disease. Percentages are column percentages. Genotyping succeeded in 1226 (100%) and 1224 (99.8%) COPACETIC participants, respectively for rs1051730 and rs8034191.</p

Linkage Disequilibrium Map for the COPD-associated variants in the 15q24/25 region.

<p>Red corresponds to r2≥0.8. Values for D′ are included in the text of boxes. The genomic positions were retrieved from the NCBI dbSNP identifier (NCBI Human genome Build 36 location). The CHRNA 5 gene variant, rs55853698, is not present in the HapMap <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053219#pone.0053219-Altshuler1" target="_blank">[21]</a>.</p

Baseline characteristics for the LEUVEN group according to rs1051730 and rs8034191 genotypes.

<p>Genotyping succeeded in 874 (99%) and 883 (100%) LEUVEN participants, respectively for rs1051730 and rs8034191.</p

DataSheet_1_Severe COVID-19 patients display hyper-activated NK cells and NK cell-platelet aggregates.pdf

COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.</p