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BackgroundThe molecular etiology of thyroid carcinoma (TC) and other thyroid diseases which may present malignant precursor lesions is not fully explored yet. The purpose of this study was to estimate frequency, type and clinicopathological value of BRAF exon 15 mutations in different types of cancerous and non-cancerous thyroid lesions originating in an ethnically diverse population.MethodsBRAF exon 15 was sequenced in 381 cases of thyroid lesions including Hashimoto´s thyroiditis, nodular goiters, hyperplastic nodules, follicular adenomas (FA), papillary TC (PTC), follicular variant PTC (FVPTC), microcarcinomas of PTC (micro PTC; tumor size ≤ 1 cm), follicular TC (FTC), and non-well differentiated TC (non-WDTC).ResultsWe identified BRAF mutations in one of 69 FA, 72 of 115 (63%) PTC, seven of 42 (17%) FVPTC, 10 of 56 (18%) micro PTC, one of 17 (6%) FTC, and one of eight (13%) non-WDTC. Most of the cases showed the common V600E mutation. One case each of PTC, FVPTC, and FTC harbored a K601E mutation. A novel BRAF mutation was identified in a FA leading to deletion of threonine at codon 599 (p.T599del). A rare 3-base pair insertion was detected in a stage III PTC resulting in duplication of threonine at codon 599 (p.T599dup). Patients with PTC harboring no BRAF mutation (BRAFwt) were on average younger than those with a BRAF mutation (BRAFmut) in the PTC (36.6 years vs. 43.8 years). Older age (≥ 45 years) in patients with PTC was significantly associated with tumor size ≥ 4 cm (P = 0.018), vessel invasion (P = 0.004), and distant metastasis (P = 0.001). Lymph node (LN) involvement in PTC significantly correlated with tumor size (P = 0.044), and vessel invasion (P = 0.013). Of notice, taken the whole TC group, family history of thyroid disease positively correlated with capsular invasion (P = 0.025).ConclusionsOlder age is manifold associated with unfavorable tumor markers in our series. The K601E identified in a PTC, FVPTC, and FTC seems to be more distributed among different histological types of TC than previously thought. The T599del is a yet undescribed mutation and the rare T599dup has not been reported as a mutation in PTC so far.

BackgroundFollicular variant of papillary thyroid carcinoma (FVPTC) and follicular adenoma (FA) are histologically closely related tumors and differential diagnosis remains challenging. RNA expression profiling is an established method to unravel molecular mechanisms underlying the histopathology of diseases.MethodsBRAF mutational status was established by direct sequencing the hotspot region of exon 15 in six FVPTCs and seven FAs. Whole-transcript arrays were employed to generate expression profiles in six FVPTCs, seven FAs and seven normal thyroid tissue samples. The threshold of significance for differential expression on the gene and exon level was a p-value with a false discovery rate (FDR) < 0.05 and a fold change cutoff > 2. Two dimensional average linkage hierarchical clustering was generated using differentially expressed genes. Network, pathway, and alternative splicing utilities were employed to interpret significance of expression data on the gene and exon level.ResultsExpression profiling in FVPTCs and FAs, all of which were negative for a BRAF mutation, revealed 55 transcripts that were significantly differentially expressed, 40 of which were upregulated and 15 downregulated in FVPTCs vs. FAs. Amongst the most significantly upregulated genes in FVPTCs were GABA B receptor, 2 (GABBR2), neuronal cell adhesion molecule (NRCAM), extracellular matrix protein 1 (ECM1), heparan sulfate 6-O-sulfotransferase 2 (HS6ST2), and retinoid X receptor, gamma (RXRG). The most significantly downregulated genes in FVPTCs included interaction protein for cytohesin exchange factors 1 (IPCEF1), G protein-coupled receptor 155 (GPR155), Purkinje cell protein 4 (PCP4), chondroitin sulfate N-acetylgalactosaminyltransferase 1 (CSGALNACT1), and glutamate receptor interacting protein 1 (GRIP1). Alternative splicing analysis detected 87 genes, 52 of which were also included in the list of 55 differentially expressed genes. Network analysis demonstrated multiple interactions for a number of differentially expressed molecules including vitamin D (1,25- dihydroxyvitamin D3) receptor (VDR), SMAD family member 9 (SMAD9), v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT), and RXRG.ConclusionsThis is one of the first studies using whole-transcript expression arrays to compare expression profiles between FVPTCs and FAs. A set of differentially expressed genes has been identified that contains valuable candidate genes to differentiate both histopathologically related tumor types on the molecular level.

We have recently shown the increased mRNA expression of interleukin (IL)-4 and IL-13 in sinus biopsies from allergic subjects with chronic sinusitis (ACS), whereas only IL-13 mRNA was elevated in biopsies obtained from nonallergic subjects with chronic sinusitis (NCS). In the lymph nodes and spleen, these cytokines may promote IgE production through transcriptional activation of the germline IgE heavy chain promoter, an event which precedes immunoglobulin isotype switching to IgE in B cells. We hypothesized that local expression of IL-4 and/or IL-13 might act by inducing germline IgE heavy chain transcript expression locally in the sinus mucosa of chronic sinusitis patients. Mucosal sinus biopsies were obtained from 13 patients with ACS, 12 subjects with NCS, and 11 normal control individuals. The numbers of B cells in the sinus mucosa were studied by immunocytochemistry with anti-CD20 monoclonal antibodies. In situ hybridization was performed using antisense radiolabeled riboprobes complementary to the IgE epsilon -heavy chain germline (Iepsilon) and heavy chain constant region (Cepsilon) gene transcripts. Riboprobes specific for the IgG gamma-heavy chain constant region (Cgamma) were used as an isotype control. Immunocytochemical analysis indicated augmented numbers of CD20-positive B cells in the biopsies obtained from ACS patients compared with NCS subjects (P < 0.05) and normal control subjects (P < 0.01). Statistically significant increases were observed in the numbers of cells expressing Iepsilon and Cepsilon transcripts in the sinus mucosa of ACS patients compared with those with NCS (P < 0. 001) and normal controls (P < 0.001), while Cgamma RNA expression did not differ significantly between the groups. In three randomly selected ACS biopsies, 92-100% of cells expressing Cepsilon transcripts and 100% of Iepsilon RNA-positive cells coexpressed CD20 immunoreactivity. Cells expressing Cepsilon transcripts were also significantly increased in NCS compared with normal controls (P < 0. 05). The results of this study suggest that local IgE class switching occurs in the pathogenesis of ACS and that ACS and NCS are both associated with increased expression of Cepsilon transcripts.

Laminar composites have widespread applications in the automotive and aircraft industry. This research was aimed to investigate the suitability of fused deposition modeling to produce multi-material laminar composites. Composites comprising of two dissimilar laminates, named as hybrid composites, were printed from acrylonitrile butadiene styrene filament and carbon fiber-reinforced polylactic acid filament (a composite filament) by varying different printing parameters. Tensile tests were conducted to examine the mechanical performance of the produced composite sheet. A detailed analysis of the results revealed that a high ultimate tensile strength is primarily achieved by setting low values of printing speed, layer height, and clad ratio while high elongation is obtained by employing low printing speed, medium layer height, and high clad ratio. The optimum printing conditions were sought out through desirability function with an objective to simultaneously enhance all the considered properties. Further, the composite sheet exhibited a reasonably good combination of tensile properties as compared to its monolithic constituent sheets. Based on the results, it is concluded that the bi-material laminating approach employed herein can produce printed structures with desired properties.