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Unlabelled: Onset and correction of hypernatraemia constitute major hypertonic stresses for mammalian cells. Cells respond by activating specific osmoregulatory mechanisms allowing to preserve their volume and to adapt to their new environmental conditions. These processes have major implications in the management of hypernatraemia. In particular, cells chronically exposed to hypertonic conditions progressively accumulate organic osmolytes to maintain optimal intracellular electrolyte concentrations. During treatment of hypernatraemia, elevated intracellular organic osmotic content exposes cells to cellular oedema if sodium concentrations are rapidly corrected. In addition, circulating ions equilibrate slowly across the blood-brain barrier during acute changes in plasma osmolality. This can generate major brain water shifts and severe cerebral lesions related to brain shrinking or cerebral oedema.Conclusion: The basic mechanisms involved in brain ion and water transport are reviewed. A proper understanding of these processes is essential to develop appropriate treatment strategies in managing children with hypernatraemia. [ABSTRACT FROM AUTHOR]

Abstract Background In-depth proteomics analyses of tumors are frequently biased by the presence of blood components and stromal contamination, which leads to large experimental variation and decreases the proteome coverage. We have established a reproducible method to prepare freshly collected lung tumors for proteomics analysis, aiming at tumor cell enrichment and reduction of plasma protein contamination. We obtained enriched tumor-cell suspensions (ETS) from six lung cancer cases (two adenocarcinomas, two squamous-cell carcinomas, two large-cell carcinomas) and from two normal lung samples. The cell content of resulting ETS was evaluated with immunocytological stainings and compared with the histologic pattern of the original specimens. By means of a quantitative mass spectrometry-based method we evaluated the reproducibility of the sample preparation protocol and we assessed the proteome coverage by comparing lysates from ETS samples with the direct lysate of corresponding fresh-frozen samples. Results Cytological analyses on cytospin specimens showed that the percentage of tumoral cells in the ETS samples ranged from 20% to 70%. In the normal lung samples the percentage of epithelial cells was less then 10%. The reproducibility of the sample preparation protocol was very good, with coefficient of variation at the peptide level and at the protein level of 13% and 7%, respectively. Proteomics analysis led to the identification of a significantly higher number of proteins in the ETS samples than in the FF samples (244 vs 109, respectively). Albumin and hemoglobin were among the top 5 most abundant proteins identified in the FF samples, showing a high contamination with blood and plasma proteins, whereas ubiquitin and the mitochondrial ATP synthase 5A1 where among the top 5 most abundant proteins in the ETS samples. Conclusion The method is feasible and reproducible. We could obtain a fair enrichment of cells but the major benefit of the method was an effective removal of contaminants from red blood cells and plasma proteins resulting in larger proteome coverage compared to the direct lysis of frozen samples. This sample preparation method may be successfully implemented for the discovery of lung cancer biomarkers on tissue samples using mass spectrometry-based proteomics.

Abstract L-acetylcarnitine (LAC), a drug utilized for the treatment of neuropathic pain in humans, has been shown to induce analgesia in rodents by up-regulating the expression of metabotropic glutamate receptor 2 (mGlu2) in dorsal root ganglia (DRG). We now report that LAC-induced upregulation of mGlu2 expression in DRG cultures involves transcriptional activation mediated by nuclear factor-kappaB (NF-κB). A single application of LAC (250 μM) to DRG cultures induced a transient increase in mGlu2 mRNA, which was observable after 1 hour and was no longer detectable after 1 to 4 days. In contrast, LAC treatment had no effect on mGlu3 mRNA expression. Pharmacological inhibition of NF-κB binding to DNA by caffeic acid phenethyl ester (CAPE) (2.5 μg/ml for 30 minutes) reduced the constitutive expression of mGlu2 and mGlu3 mRNA after 1–4 days and reduced the constitutive expression of mGlu2/3 protein at 4 days. This evidence combined with the expression of p65/RelA and c-Rel in DRG neurons indicated that expression of mGlu2 and mGlu3 is endogenously regulated by the NF-κB family of transcription factors. Consistent with this idea, the transient increase in mGlu2 mRNA induced by LAC after 1 hour was completely suppressed by CAPE. Furthermore, LAC induced an increase in the acetylation of p65/RelA, a process that enhances the transcriptional activity of p65/RelA. These results are consistent with the hypothesis that LAC selectively induces the expression of mGlu2 by acting as a donor of acetyl groups, thus enhancing the activity of the NF-κB family of transcription factors. Accordingly, we show that carnitine, which has no effect on pain thresholds, had no effect on p65/RelA acetylation and did not enhance mGlu2 expression. Taken together, these results demonstrate that expression of mGlu2 and mGlu3 mRNA is regulated by the NF-κB transcriptional machinery, and that agents that increase acetylation and activation of NF-κB transcription factors might induce analgesia via upregulation of mGlu2 in DRG neurons.

Background: Rearrangement in anaplastic lymphoma kinase ( ALK ) occurs in 4-7% of non-small cell lung cancer (NSCLC) cases. Despite improved survival with tyrosine kinase inhibitors (TKIs), treatment resistance remains challenging. This retrospective study analyzed advanced ALK-positive NSCLC patients, focusing on clinical aspects, treatments, resistance, and outcomes., Methods: Patients diagnosed between January 2009 and December 2021 who received at least one ALK-TKI line at the Karolinska University Hospital, were included. We evaluated crizotinib or 2 nd generation ALK-TKI effectiveness in first-line treatment and lorlatinib in subsequent lines. Overall survival (OS) was defined as from the date of advanced lung cancer diagnosis until the date of last follow-up (April 22, 2022) or the date of death from any cause. Progression-free survival (PFS), from the date of starting ALK-TKI until the date of progression, death, or last follow-up. Resistance mechanisms were assessed through re-biopsies utilizing next-generation sequencing (NGS)., Results: Out of 160 eligible patients, 10 were excluded. Median follow-up was 54.0 months from diagnosis and 45.0 months from initial ALK-TKI treatment. Crizotinib showed a median PFS of 8.0 months and a median OS of 35.0 months. Second generation ALK-TKIs demonstrated a median PFS of 52.0 months [OS was not reached (NR)]. Overall, the median OS was 65.0 months. Poor prognostic factors included male sex, thromboembolism, crizotinib treatment, and chronic obstructive pulmonary disease (COPD)/asthma. Rebiopsies in 18 cases revealed secondary ALK mutations in 8 patients, correlating with a shorter median PFS in subsequent ALK-TKI treatment (1.0 vs. 7.0 months)., Conclusions: This comprehensive study, spanning over a decade, provides crucial insights into the clinical characteristics, treatment patterns, and resistance mechanisms of advanced ALK-positive NSCLC, where median OS exceeds 5 years. Re-biopsies during treatment are essential for advancing our understanding of resistance mechanisms and the tumor dynamics evolving during ALK-TKI therapy., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-24-396/coif). G.T. reports honoraria from MSD, Roche AB for lectures and reports paid participation at ELCC 2023 from Roche AB. K.V. is supported for salary and other project related costs by the indicated private funding bodies or region grants Stockholm Cancer Society (contract No. 221383), Stockholm County Council (contract Nos. 909121, 750032, and FoUI-966345), and Erling Persson Family Foundation. R.L. reports grant from the Swedish Cancer Society (No. CAN2021/1469), the Stockholm Cancer Society (No. 221212), the Stockholm County Council (contract Nos. FoUI-966345, 909121, and 750032), the Erling Persson Family Foundation, and the Swedish Foundation for Strategic Research (No. SIP21-0106). S.E. reports grants from the Swedish Cancer Society (grant No. CAN2023/2919), the King Gustaf V Jubilee Fund (grant No. 204053), the Sjöberg Foundation (No. 2022-2024), and the Stockholm County Council (No. 987911). The other authors have no conflicts of interest to declare., (2024 AME Publishing Company. All rights reserved.)

Introduction: Programmed death ligand - 1 (PD-L1) expression is a well-established predictive biomarker for immunotherapy in non-small cell lung cancer (NSCLC). Programmed death - 1 (PD-1) serves as the target protein to PD-L1 and their interaction serves as a crucial pathway for immune evasion. This study aimed to investigate the expression pattern of PD-1 on Tumor-infiltrating lymphocytes (TILs) in early-stage NSCLC, and its potential role as prognostic biomarker., Materials & Methods: PD-1 was evaluated in 474 surgical resected early-stage NSCLC specimens, using Tissue microarray and immunohistochemical staining. Expression was scored as negative (<1%) or positive. Positive PD-1 expression was further divided into low (<10%) and high (≥10%). None of the patients had received treatment with PD-1/PD-L1 inhibitors., Results: PD-1 expression ≥1% in TILs was observed in 83.5% of cases and was associated with pT stage (p=0.02), grade 3 (p=0.004), and adenocarcinoma subtype (p=0.05). Individuals with high PD-1 expression (≥10%) experienced reduced 10-year overall survival (Log-Rank test = 0.005). In addition, high PD-1 expression emerged as an independent factor associated with reduced survival on multivariate analysis (HR: 1.328 (95% CI: 1.074-1.641)., Conclusions: Patients with early-stage NSCLC who exhibited PD-1 expression of ≥10% on TILs had an unfavorable 10-year OS rate. These findings indicate that elevated PD-1 expression on TILs can be associated with immune evasion during the early stages of malignancy evolution in the NSCLC setting and further research is required to further delineate the role of PD-1/PD-L1 pathway on tumor immune senescence. These results underline the potential role of PD-1/PD-L1 inhibitors in the treatment of early-stage NSCLC., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dan, Aricak, Rounis, Montero-Fernandez, Guijarro, Ekman, Ortiz-Villalón and De Petris.)

The potential of real-world data to inform clinical trial design and supplement control arms has gained much interest in recent years. The most common approach relies on reproducing control arm outcomes by matching real-world patient cohorts to clinical trial baseline populations. However, recent studies pointed out that there is a lack of replicability, generalisability, and consensus. In this article, we propose a novel approach that aims to explore and examine these discrepancies by concomitantly investigating the impact of selection criteria and operations on the measurements of outcomes from the patient data. We tested the approach on a dataset consisting of small-cell lung cancer patients receiving platinum-based chemotherapy regimens from a real-world data cohort (n = 223) and six clinical trial control arms (n = 1224). The results showed that the discrepancy between real-world and clinical trial data potentially depends on differences in both patient populations and operational conditions (e.g., frequency of assessments, and censoring), for which further investigation is required. Discovering and accounting for confounders, including hidden effects of differences in operations related to the treatment process and clinical trial study protocol, would potentially allow for improved translation between clinical trials and real-world data. Continued development of the method presented here to systematically explore and account for these differences could pave the way for transferring learning across clinical studies and developing mutual translation between the real-world and clinical trials to inform clinical study design., (© 2024 The Author(s). Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Detection of analytes using streaming current has previously been explored using both experimental approaches and theoretical analyses of such data. However, further developments are needed for establishing a viable microchip that can be exploited to deliver a sensitive, robust, and scalable biosensor device. In this study, we demonstrated the fabrication of such a device on silicon wafer using a scalable silicon microfabrication technology followed by characterization and optimization of this sensor for detection of small extracellular vesicles (sEVs) with sizes in the range of 30 to 200 nm, as determined by nanoparticle tracking analyses. We showed that the sensitivity of the devices, assessed by a common protein-ligand pair and sEVs, significantly outperforms previous approaches using the same principle. Two versions of the microchips, denoted as enclosed and removable-top microchips, were developed and compared, aiming to discern the importance of high-pressure measurement versus easier and better surface preparation capacity. A custom-built chip manifold allowing easy interfacing with standard microfluidic connections was also constructed. By investigating different electrical, fluidic, morphological, and fluorescence measurements, we show that while the enclosed microchip with its robust glass-silicon bonding can withstand higher pressure and thus generate higher streaming current, the removable-top configuration offers several practical benefits, including easy surface preparation, uniform probe conjugation, and improvement in the limit of detection (LoD). We further compared two common surface functionalization strategies and showed that the developed microchip can achieve both high sensitivity for membrane protein profiling and low LoD for detection of sEV detection. At the optimum working condition, we demonstrated that the microchip could detect sEVs reaching an LoD of 10 4 sEVs/mL (when captured by membrane-sensing peptide (MSP) probes), which is among the lowest in the so far reported microchip-based methods.

Process mining is a relatively new method that connects data science and process modelling. In the past years a series of applications with health care production data have been presented in process discovery, conformance check and system enhancement. In this paper we apply process mining on clinical oncological data with the purpose of studying survival outcomes and chemotherapy treatment decision in a real-world cohort of small cell lung cancer patients treated at Karolinska University Hospital (Stockholm, Sweden). The results highlighted the potential role of process mining in oncology to study prognosis and survival outcomes with longitudinal models directly extracted from clinical data derived from healthcare.

The ROR1 receptor tyrosine kinase is expressed in embryonic tissues but is absent in normal adult tissues. ROR1 is of importance in oncogenesis and is overexpressed in several cancers, such as NSCLC. In this study, we evaluated ROR1 expression in NSCLC patients (N = 287) and the cytotoxic effects of a small molecule ROR1 inhibitor (KAN0441571C) in NSCLC cell lines. ROR1 expression in tumor cells was more frequent in non-squamous (87%) than in squamous (57%) carcinomas patients, while 21% of neuroendocrine tumors expressed ROR1 ( p = 0.0001). A significantly higher proportion of p53 negative patients in the ROR1 + group than in the p53 positive non-squamous NSCLC patients ( p = 0.03) was noted. KAN0441571C dephosphorylated ROR1 and induced apoptosis (Annexin V/PI) in a time- and dose-dependent manner in five ROR1 + NSCLC cell lines and was superior compared to erlotinib (EGFR inhibitor). Apoptosis was confirmed by the downregulation of MCL-1 and BCL-2, as well as PARP and caspase 3 cleavage. The non-canonical Wnt pathway was involved. The combination of KAN0441571C and erlotinib showed a synergistic apoptotic effect. KAN0441571C also inhibited proliferative (cell cycle analyses, colony formation assay) and migratory (scratch wound healing assay) functions. Targeting NSCLC cells by a combination of ROR1 and EGFR inhibitors may represent a novel promising approach for the treatment of NSCLC patients.

Background: Targeted therapy with tyrosine kinases inhibitors (TKIs) against epidermal growth factor receptor (EGFR) is part of routine clinical practice for EGFR mutant advanced non-small cell lung cancer (NSCLC) patients. These patients eventually develop resistance, frequently accompanied by a gatekeeper mutation, T790M. Osimertinib is a third-generation EGFR TKI displaying potency to the T790M resistance mutation. Here we aimed to analyze if exosomal RNAs, isolated from longitudinally sampled plasma of osimertinib-treated EGFR T790M NSCLC patients, could provide biomarkers of acquired resistance to osimertinib., Methods: Plasma was collected at baseline and progression of disease from 20 patients treated with osimertinib in the multicenter phase II study TKI in Relapsed EGFR-mutated non-small cell lung cancer patients (TREM). Plasma was centrifuged at 16,000 g followed by exosomal RNA extraction using Qiagen exoRNeasy kit. RNA was subjected to transcriptomics analysis with Clariom D., Results: Transcriptome profiling revealed differential expression [log2(fold-change) >0.25, false discovery rate (FDR) P<0.15, and P(interaction) >0.05] of 128 transcripts. We applied network enrichment analysis (NEA) at the pathway level in a large collection of functional gene sets. This overall enrichment analysis revealed alterations in pathways related to EGFR and PI3K as well as to syndecan and glypican pathways (NEA FDR <3×10 -10 ). When applied to the 40 individual, sample-specific gene sets, the NEA detected 16 immune-related gene sets (FDR <0.25, P(interaction) >0.05 and NEA z-score exceeding 3 in at least one sample)., Conclusions: Our study demonstrates a potential usability of plasma-derived exosomal RNAs to characterize molecular phenotypes of emerging osimertinib resistance. Furthermore, it highlights the involvement of multiple RNA species in shaping the transcriptome landscape of osimertinib-refractory NSCLC patients., Competing Interests: Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-236/coif). The authors have no conflicts of interest to declare., (2022 Translational Lung Cancer Research. All rights reserved.)

In recent studies, small cell lung cancer (SCLC) treatment guidelines based on Veterans' Administration Lung Study Group limited/extensive disease staging and resulted in broad and inseparable prognostic subgroups. Evidence suggests that the eight versions of tumor, node, and metastasis (TNM) staging can play an important role to address this issue. The aim of the present study was to improve the detection of prognostic subgroups from a real-word data (RWD) cohort of patients and analyze their patterns using a development pipeline with thoracic oncologists and machine learning methods. The method detected subgroups of patients informing unsupervised learning (partition around medoids) including the impact of covariates on prognosis (Cox regression and random survival forest). An analysis was carried out using patients with SCLC (n = 636) with stage IIIA-IVB according to TNM classification. The analysis yielded k = 7 compacted and well-separated clusters of patients. Performance status (Eastern Cooperative Oncology Group-Performance Status), lactate dehydrogenase, spreading of metastasis, cancer stage, and CRP were the baselines that characterized the subgroups. The selected clustering method outperformed standard clustering techniques, which were not capable of detecting meaningful subgroups. From the analysis of cluster treatment decisions, we showed the potential of future RWD applications to understand disease, develop individualized therapies, and improve healthcare decision making., (© 2022 The Authors. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Treatment with the tyrosine kinase inhibitor (TKI) osimertinib is the standard of care for non-small cell lung cancer (NSCLC) patients with activating mutations in the epidermal growth factor receptor (EGFR). Osimertinib is also used in T790M-positive NSCLC that may occur de novo or be acquired following first-line treatment with other EGFR TKIs (i.e., gefitinib, erlotinib, afatinib, or dacomitinib). However, patients treated with osimertinib have a high risk of developing resistance to the treatment. A substantial fraction of the mechanisms for resistance is unknown and may involve RNA and/or protein alterations. In this study, we investigated the full transcriptome of parental and osimertinib-resistant cell lines, revealing 131 differentially expressed genes. Knockdown screening of the genes upregulated in resistant cell lines uncovered eight genes to partly confer resistance to osimertinib. Among them, we detected the expression of Ras-related protein Rab-32 (RAB32) and thrombospondin 1 (THBS1) in plasmas sampled at baseline and at disease progression from EGFR-positive NSCLC patients treated with osimertinib. Both genes were upregulated in progression samples. Moreover, we found that knockdown of RAB32 and THBS1 reduced the expression of phosphorylated focal adhesion kinase (FAK). Combination of osimertinib with a FAK inhibitor resulted in synergistic toxicity in osimertinib-resistant cells, suggesting a potential therapeutic drug combination for overcoming resistance to osimertinib in NSCLC patients.