Changes in the use of antiseizure medications in children and adolescents in Norway, 2009-2018.

BACKGROUND: The use of antiseizure medications (ASMs) in the pediatric population is poorly studied. The purpose of this study was to investigate changes in the use of ASMs in children and adolescents compared to adults, and to elucidate safety considerations of certain drugs. METHOD: In this population-based pharmacoepidemiological study we used the Norwegian Prescription Database (NorPD), 2009-2018. The use of ASMs is presented as 1-year prevalence per 1000: number of ASM users in a year * 1000 / number of inhabitants that year. Variables included predetermined 5-year age groups, gender, ASMs, diagnosis-specific reimbursement codes, user, and population numbers. Selected ASMs used for specific indications or subgroups included ethosuximide, sulthiame, rufinamide, stiripentol, and clobazam. Gender differences in the use of valproate was examined due to safety considerations in girls/women. RESULTS: The total number of ASM users (all indications) for the age groups 0-19 and 20-59 years was 5807 and 47,481 respectively in 2009, and 5906 and 61,447 respectively in 2018. The 1-year prevalence for children/adolescents (0-19 years) using ASMs in epilepsy remained stable from 2008 to 2018, 4.3-4.2/1000 inhabitants, as compared to 8.2-7.6/1000 in adults (20-59 years). Valproate, lamotrigine, and levetiracetam were the three most used ASMs in epilepsy in children/adolescents, similar to adults. The selected ASMs were mainly used in children/ adolescents, accounting for 0.74/1000 in 2018 versus 0.17/1000 in adults. A significant increase was seen for sulthiame (8-fold), ethosuximide (4-fold), clobazam (3-fold), and stiripentol (2-fold). The use of ASMs in non-epilepsy indications was limited and stable (17% in 2018); mainly lamotrigine in psychiatry in adolescents (15-19 years). This finding was in contrast to extensive non-epilepsy use in adults (71% in 2018). CONCLUSION: Changes in the use of ASMs in children/adolescents differ as compared to adults, most notably extensive and increasing use of selected ASMs and limited non-epilepsy. This is an important part of pharmacovigilance and patient safety evaluations.

Nationwide Study of Neuropsychiatric Comorbidity and Medicines Use in Children With Autism Spectrum Disorder in Norway.

Purpose: Autism spectrum disorder (ASD) has a high rate of comorbidity. While many children with ASD are exposed to psychotropic medicines, their efficacy and safety in these patients are unclear. There is a need for more detailed knowledge on which medicines are most commonly used and for which disorders. We aimed to investigate (a) prevalence and incidence rate of ASD among Norwegian children, and further, among newly diagnosed ASD children in 2014, study the (b) co-occurrence of neuropsychiatric disorders, (c) use of psychotropic drugs, and (d) the relationship between co-occurring diagnoses and use of psychotropic drugs. Method: Nationwide registry-based study of children 2-17 years old in Norway. Results: The ASD prevalence was 0.76% and the incidence rate was 0.12% in 2014. Of the children who received an initial ASD diagnosis in 2014 (n = 1,234), 64.8% had one or more co-occurring neuropsychiatric diagnosis. Psychotropic medication use was moderate (~20% used stimulants or hypnotics) in general, and low in children without comorbidity (nearly only hypnotics). There was a good accordance between co-occurring diagnoses and indication for the prescribed medications. Conclusions: Children with newly diagnosed ASD mainly received psychotropic drugs to treat co-occurring neuropsychiatric conditions.

Transcription factor PAX6 as a novel prognostic factor and putative tumour suppressor in non-small cell lung cancer.

Lung cancer is the leading cause of cancer deaths. Novel predictive biomarkers are needed to improve treatment selection and more accurate prognostication. PAX6 is a transcription factor with a proposed tumour suppressor function. Immunohistochemical staining was performed on tissue microarrays from 335 non-small cell lung cancer (NSCLC) patients for PAX6. Multivariate analyses of clinico-pathological variables and disease-specific survival (DSS) was carried out, and phenotypic changes of two NSCLC cell lines with knockdown of PAX6 were characterized. While PAX6 expression was only associated with a trend of better disease-specific survival (DSS) (p = 0.10), the pN+ subgroup (N = 103) showed significant correlation between high PAX6 expression and longer DSS (p = 0.022). Median survival for pN + patients with high PAX6 expression was 127.4 months, versus 22.9 months for patients with low PAX6 expression. In NCI-H661 cells, knockdown of PAX6 strongly activated serum-stimulated migration. In NCI-H460 cells, PAX6 knockdown activated anchorage-independent growth. We did not observe any significant effect of PAX6 on proliferation in either of cell lines. Our findings strongly support the proposition of PAX6 as a valid and positive prognostic marker in NSCLC in node-positive patients. There is a need for further studies, which should provide mechanistical explanation for the role of PAX6 in NSCLC.

Expression and function of the miR-143/145 cluster in vitro and in vivo in human breast cancer.

MicroRNAs (miRNAs) are small non-coding RNAs that function as post-transcriptional regulators of gene expression and are dysregulated in cancer. Studies of miRNAs to explore their potential as diagnostic and prognostic markers are of great scientific interest. Here, we investigate the functional properties and expression of the miR-143/145 cluster in breast cancer (BC) in vitro and in vivo. The ER positive MCF7, the HER2 positive SK-BR-3, and the triple negative cell line MDA-MB-231 were used to assess cell proliferation and cell invasion. Expression of miRNA in 108 breast cancers in the Norwegian Women and Cancer Study and 44 benign tissue controls were analyzed by microarray and validated by RT-PCR. Further, in situ hybridization (ISH) was used to study the cellular and subcellular distribution of the miRNAs. In vitro, miR-143 promoted proliferation of MCF7 and MDA-MB-231 cells, whereas miR-145 and the cotransfection of both miRNAs inhibited proliferation in all three cell lines. The cells' invasive capacity was reduced after transfection and cotransfection of the miRNAs. In line with the tumor suppressive functions in vitro, the expression of miR-143 and miR-145 was lower in malignant compared to benign breast tissue, and lower in the more aggressive tumors with higher tumor grade, loss of ER and the basal-like phenotype. ISH revealed miR-143 to be cytoplasmatic and predominantly expressed in luminal cells in benign tissue, whilst miR-145 was nuclear and with strong staining in myoepithelial cells. Both miRNAs were present in malignant epithelial cells and stromal fibroblasts in BC. This study demonstrates that miR-143 and -145 have functional properties and expression patterns typical for tumor suppressors, but the function is influenced by cellular factors such as cell type and miRNA cotransfection. Further, the nuclear functions of miR-145 should be explored for a more complete understanding of the complexity of miRNA regulation and function in BC.

Homeostatic regulation in physiological systems: A versatile Ansatz.

A generic modelling formalism is described for homeostatic dynamics in physiological systems. The method is particularly suited where the peripheral, physiological system itself is well-characterised, but the details of the central, regulatory component (the nervous and endocrine systems) have not necessarily been characterised in full detail. The method is applied to temperature regulation in Cardinalis cardinalis, C. sinuatus, Lepus alleni, and Passer domesticus, and furthermore to hydromineral regulation in Lymnaea stagnalis. These case studies demonstrate that the method allows a comprehensive analysis and integration of the available data and is capable of furnishing physiologically relevant predictions. We discuss the method in relation to optimal control theory as well as more conventional modelling approaches.

Prognostic impact of CXCL16 and CXCR6 in non-small cell lung cancer: combined high CXCL16 expression in tumor stroma and cancer cells yields improved survival.

BACKGROUND: The chemokine CXCL16 and its receptor CXCR6 are expressed by a variety of immune cells and have been shown to influence angiogenesis. The expression of CXCR6 and CXCL16 has been examined in numerous human cancers; however no studies have yet investigated their influence on prognosis in non-small cell lung cancer (NSCLC). We aimed to explore their prognostic significance in NSCLC, in addition to examining associations with previously investigated markers. METHODS: Resected tumor tissue from 335 consecutive unselected stage I-IIIA NSCLC patients (1990-2005) were collected. Immunohistochemistry was used to evaluate the expression of CXCR6 and CXCL16 on tissue microarrays. In vitro, NSCLC cells (NCI-H460, A549 cells) were transfected with CXCL16 siRNA to examine effects on proliferation. RESULTS: In univariate analysis, ↑ stromal cell CXCL16 expression was a significant positive prognostic factor (P = 0.016). CXCR6 was expressed in cancer cells, but did not show any prognostic impact. In the multivariate analysis, combined ↑cancer, and ↑stromal cell CXCL16 expression was an independent positive prognostic factor when compared to ↓stromal and ↓cancer cell expression (HR: 0.42; 95 % CI: 0.20-0.88; P = 0.022). Knockdown of CXCL16 by siRNA resulted in accelerated proliferation of NSCLC cell lines. CONCLUSION: We have shown that combined ↑cancer and ↑stromal cell CXCL16 expression is an independent positive prognostic factor in NSCLC. Further studies are warranted to elucidate the biological mechanism underlying this finding.

Stromal CD8+ T-cell Density­A Promising Supplement to TNM Staging in Non-Small Cell Lung Cancer.

PURPOSE: Immunoscore is a prognostic tool defined to quantify in situ immune cell infiltrates, which appears to be superior to the tumor-node-metastasis (TNM) classification in colorectal cancer. In non-small cell lung cancer (NSCLC), no immunoscore has been established, but in situ tumor immunology is recognized as highly important. We have previously evaluated the prognostic impact of several immunological markers in NSCLC, yielding the density of stromal CD8(+) tumor-infiltrating lymphocytes (TIL) as the most promising candidate. Hence, we validate the impact of stromal CD8(+) TIL density as an immunoscore in NSCLC. EXPERIMENTAL DESIGN: The prognostic impact of stromal CD8(+) TILs was evaluated in four different cohorts from Norway and Denmark consisting of 797 stage I-IIIA NSCLC patients. The Tromso cohort (n = 155) was used as training set, and the results were further validated in the cohorts from Bodo (n = 169), Oslo (n = 295), and Denmark (n = 178). Tissue microarrays and clinical routine CD8 staining were used for all cohorts. RESULTS: Stromal CD8(+) TIL density was an independent prognostic factor in the total material (n = 797) regardless of the endpoint: disease-free survival (P < 0.001), disease-specific survival (P < 0.001), or overall survival (P < 0.001). Subgroup analyses revealed significant prognostic impact of stromal CD8(+) TIL density within each pathologic stage (pStage). In multivariate analysis, stromal CD8(+) TIL density and pStage were independent prognostic variables. CONCLUSIONS: Stromal CD8(+) TIL density has independent prognostic impact in resected NSCLC, adds prognostic impact within each pStage, and is a good candidate marker for establishing a TNM-Immunoscore.

The prognostic role of progesterone receptor expression in non-small cell lung cancer patients: Gender-related impacts and correlation with disease-specific survival.

PURPOSE: Progesterone has been shown to impact the development of hormone-sensitive cancers, such as breast and ovarian cancers. Emerging evidence has revealed a possible role of progesterone in the tumorigenesis of other cancers, including lung cancer. Herein, we aimed to elucidate the prevalence and prognostic significance of progesterone receptor (PR) expression in non-small cell lung cancer (NSCLC) tissue. EXPERIMENTAL: Tumor tissue samples were collected from our patient cohort consisting of 335 NSCLC patients with stage I-IIIA disease. Tissue microarrays (TMAs) were constructed, and immunohistochemical (IHC) analyses were performed to evaluate the PR expression in the tumor epithelial and stromal compartments. RESULTS: In a univariate analysis, positive PR expression in the stromal tumor compartment (P=0.005) was significantly and independently associated with a favorable outcome for both genders. Furthermore, positive PR expression in tumor epithelial cells (P=0.003) correlated with a poor prognosis for female patients. In a multivariate analysis, positive PR expression in the tumor stroma (P=0.007) was an independent prognostic factor for improved disease-specific survival (DSS). Positive PR expression in tumor epithelial cells emerged as an independent prognostic factor in female patients (P=0.001) for poor DSS. CONCLUSIONS: We show that PR expression in tumor-surrounding stromal cells is associated with improved DSS for both male and female patients. Additionally, we reveal that positive PR expression in tumor epithelial cells is an independent, unfavorable prognosticator for DSS in female patients, making PR expression a potential marker for prognostic stratification in NSCLC.

High progesterone receptor expression in prostate cancer is associated with clinical failure.

BACKGROUND: Prostate cancer is a highly heterogeneous disease and one of the leading causes of mortality in developed countries. Specific prognostic and predictive markers for prostate cancer patients are still lacking. A causal relationship between androgens and the development of prostate cancer is generally considered biologically plausible, but androgens are not the sole effector in the complexity of prostate carcinogenesis. The aim of this study was to evaluate the prognostic significance of progesterone receptor in tumor tissue of T1-3N0 prostate cancer patients undergoing prostatectomy. METHODS: Tissue microarrays from 535 patients with prostate cancer were constructed. Duplicate cores of tumor cells and tumor stromal tissue from each resected specimen were extracted. Immunohistochemistry was used to evaluate the in-situ expression of progesterone receptor. RESULTS: In univariate analyses, high tumor cell density (p = 0.006) and high tumor stromal cell density level (p = 0.045) of progesterone receptor were both significantly associated with tumor progression and clinical failure. In multivariate analysis, progesterone receptor expression in tumor cells was an independent negative prognostic factor for clinical failure (HR: 2.5, 95% CI: 1.2-5.2, p = 0.012). CONCLUSION: High progesterone receptor density in tumor cells of the prostate cancer tumor is an independent negative prognostic factor for clinical failure.

Stromal expression of MiR-21 predicts biochemical failure in prostate cancer patients with Gleason score 6.

AIM: microRNAs (miRNAs) are involved in various neoplastic diseases, including prostate cancer (PCs). The aim of this study was to investigate the miRNA profile in PC tissue, to assess their association with clinicopathologic data, and to evaluate the potential of miRNAs as diagnostic and prognostic markers. MATERIALS AND METHODS: From a cohort of 535 patients submitted to radical prostatectomy (RP), a sample of 30 patients (14 patients with rapid biochemical failure (BF) and 16 patients without BF) with Gleason score 7 were analyzed. A total of 1435 miRNAs were quantified by microarray hybridization, and selected miRNAs with the highest Standard deviation (n = 50) were validated by real-time quantitative PCR (qRT-PCR). In situ hybridization (ISH) was used to evaluate the expression of miR-21. RESULTS: miR-21 was the only miR that was significantly up-regulated in the BF group (p = 0.045) miR-21 was up-regulated in patients with BF compared with non-BF group (p = 0.05). In univariate analyses, high stromal expression of miR-21 had predictive impact on biochemical failure-free survival (BFFS) and clinical failure-free survival (CFFS) (p = 0.006 and p = 0.04, respectively). In the multivariate analysis, high stromal expression of miR-21 expression was found to be an independent prognostic factor for BFFS in patients with Gleason score 6 (HR 2.41, CI 95% 1.06-5.49, p = 0.037). CONCLUSION: High stromal expression of miR-21 was associated with poor biochemical recurrence-free survival after RP. For patients with Gleason score 6, miR-21 may help predict the risk of future disease progression and thereby help select patients for potential adjuvant treatment or a more stringent follow-up.

Monocarboxylate transporters 1-4 in NSCLC: MCT1 is an independent prognostic marker for survival.

INTRODUCTION: Monocarboxylate transporters (MCTs) 1-4 are lactate transporters crucial for cancers cells adaption to upregulated glycolysis. Herein, we aimed to explore their prognostic impact on disease-specific survival (DSS) in both cancer and tumor stromal cells in NSCLC. METHODS: Tissue micro arrays (TMAs) were constructed, representing both cancer and stromal tumor tissue from 335 unselected patients diagnosed with stage I-IIIA NSCLC. Immunohistochemistry was used to evaluate the expression of MCT1-4. RESULTS: In univariate analyses; ↓ MCT1 (P = 0.021) and ↑ MCT4 (P = 0.027) expression in cancer cells, and ↑ MCT1 (P = 0.003), ↓ MCT2 (P = 0.006), ↓ MCT3 (P = 0.020) expression in stromal cells correlated significantly with a poor DSS. In multivariate analyses; ↓ MCT1 expression in cancer cells (HR: 1.9, CI 95%: 1.3-2.8, P = 0.001), ↓ MCT2 (HR: 2.4, CI 95%: 1.5-3.9, P<0.001), ↓ MCT3 (HR: 1.9, CI 95%: 1.1-3.5, P = 0.031) and ↑ MCT1 expression in stromal cells (HR: 1.7, CI 95%: 1.1-2.7, P = 0.016) were significant independent poor prognostic markers for DSS. CONCLUSIONS: We provide novel information of MCT1 as a candidate marker for prognostic stratification in NSCLC. Interestingly, MCT1 shows diverging, independent prognostic impact in the cancer cell and stromal cell compartments.

Pax6 regulates the expression of Dkk3 in murine and human cell lines, and altered responses to Wnt signaling are shown in FlpIn-3T3 cells stably expressing either the Pax6 or the Pax6(5a) isoform.

Pax6 is a transcription factor important for early embryo development. It is expressed in several cancer cell lines and tumors. In glioblastoma, PAX6 has been shown to function as a tumor suppressor. Dickkopf 3 (Dkk3) is well established as a tumor suppressor in several tumor types, but not much is known about the regulation of its expression. We have previously found that Pax6 and Pax6(5a) increase the expression of the Dkk3 gene in two stably transfected mouse fibroblast cell lines. In this study the molecular mechanism behind this regulation is looked at. Western blot and reverse transcriptase quantitative PCR (RT-qPCR) confirmed higher level of Dkk3 expression in both Pax6 and Pax6(5a) expressing cell lines compared to the control cell line. By the use of bioinformatics and electrophoretic mobility shift assay (EMSA) we have mapped a functional Pax6 binding site in the mouse Dkk3 promoter. The minimal Dkk3 promoter fragment required for transcriptional activation by Pax6 and Pax6(5a) was a 200 bp region just upstream of the transcriptional start site. Mutation of the evolutionary conserved binding site in this region abrogated transcriptional activation and binding of Pax6/Pax6(5a) to the mouse Dkk3 promoter. Since the identified Pax6 binding site in this promoter is conserved, RT-qPCR and Western blot were used to look for regulation of Dkk3/REIC expression in human cell lines. Six of eight cell lines tested showed changes in Dkk3/REIC expression after PAX6 siRNA knockdown. Interestingly, we observed that the Pax6/Pax6(5a) expressing mouse fibroblast cell lines were less responsive to canonical Wnt pathway stimulation than the control cell line when TOP/FOP activity and the levels of active ß-catenin and GSK3-ß Ser9 phosphorylation were measured after LiCl stimulation.

3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes.

Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.