Next-generation sequencing facilitates detection of the classic E13-A20 EML4-ALK fusion in an ALK-FISH/IHC inconclusive biopsy of a stage IV lung cancer patient: a case report

Background

Inhibition of the oncogenic fusion-gene EML4-ALK is a current first-line approach for patients with stage IV non-small cell lung cancer. While FISH was established as the gold standard for identifying these patients, there is accumulating evidence that other methods of detection, i.e., immunohistochemistry and next-generation sequencing (NGS), exist that may be equally successful. However, the concordance of these methods is under investigation.

Case presentation

Adding to the current literature, we here report a 56 year old female never-smoker with stage IV lung adenocarcinoma whose biopsy was IHC and FISH inconclusive but positive in NGS. Retroactive profiling of the resection specimen corroborated fusion reads obtained by NGS, FISH-positivity and showed weak ALK-positivity by IHC. Consequently, we diagnosed the case as ALK-positive rendering the patient eligible to crizotinib treatment.

Conclusions

With IHC on biopsy material only, this case would have been overlooked withholding effective therapy.

     

A Cre-loxP based system for studying horizontal gene transfer

We have previously shown that DNA can be transferred to phagocytosing cells via the uptake of apoptotic cells. We report a model system that facilitates study of antigen presentation of genes transferred specifically via horizontal gene transfer. Constructs were generated encoding the LacZ gene or the influenza A nucleoprotein silenced by a STOP sequence flanked by two loxP sites. These reporter genes were demonstrated to be silent in donor cells and become activated after phagocytosis of Cre-expressing fibroblasts or macrophages. These results provide a model system for studying the influence of horizontally transferred antigens on activation of the immune system.     

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

Exogenous Nef Is an Inhibitor of Mycobacterium tuberculosis-induced Tumor Necrosis Factor-�� Production and Macrophage Apoptosis

Human immunodeficiency virus-1 (HIV-1) impairs tumor necrosis factor-α (TNF-α)-mediated macrophage apoptosis induced by Mycobacterium tuberculosis (Mtb). HIV Nef protein plays an important role in the pathogenesis of AIDS. We have tested the hypothesis that exogenous Nef is a factor that inhibits TNF-α production/apoptosis in macrophages infected with Mtb. We demonstrate that Mtb and Nef individually trigger TNF-α production in macrophages. However, TNF-α production is dampened when the two are present simultaneously, probably through cross-regulation of the individual signaling pathways leading to activation of the TNF-α promoter. Mtb-induced TNF-α production is abrogated upon mutation of the Ets, Egr, Sp1, CRE, or AP1 binding sites on the TNF-α promoter, whereas Nef-mediated promoter activation depends only on the CRE and AP1 binding sites, pointing to differences in the mechanisms of activation of the promoter. Mtb-dependent promoter activation depends on the mitogen-activated kinase (MAPK) kinase kinase ASK1 and on MEK/ERK signaling. Nef inhibits ASK1/p38 MAPK-dependent Mtb-induced TNF-α production probably by inhibiting binding of ATF2 to the TNF-α promoter. It also inhibits MEK/ERK-dependent Mtb-induced binding of FosB to the promoter. Nef-driven TNF-α production occurs in an ASK1-independent, Rac1/PAK1/p38 MAPK-dependent, and MEK/ERK-independent manner. The signaling pathways used by Mtb and Nef to trigger TNF-α production are therefore distinctly different. In addition to attenuating Mtb-dependent TNF-α promoter activation, Nef also reduces Mtb-dependent TNF-α mRNA stability probably through its ability to inhibit ASK1/p38 MAPK signaling. These results provide new insight into how HIV Nef probably exacerbates tuberculosis infection by virtue of its ability to dampen Mtb-induced TNF-α production.     

Deceit pollination of Orchis spitzelii (Orchidaceae) on the Island of Gotland in the Baltic: a suboptimal system

Interactions between anthophilous insects and Orchis spitzelii were studied on the island of Gotland, in the Baltic Sea of southern Sweden. The plant is nectar - less and thus deceives its nectar-seeking pollinators. No specific nectar-plant model exists for mimetic resemblance, but the orchid produces an exuberant floral display. Pollinators comprise mainly bumble-bee queens ( Bombus spp.) that transport 90% of the pollinaria. The remaining pollinators are cuckoo bumble-bee females ( Psithyrus spp.). Pollinating bees make exploratory visits primarily to only a few of the lowermost flowers in inflorescences. Many-flowered individuals are more attractive and have a higher fitness in terms of fruit set. Bees quickly learn to avoid the non-rewarding orchid. Arctostaphylos uva-ursi (Ericacae) and Rubus saxatilis (Rosaceae) are the most important subsidiary nectar plants for the bees during the flowering of O. spitzelii. Phenological, morphological and functional data indicate that the anth-ecological adaptation to the main pollen vectors is suboptimal. Moreover, experiments indicate that O. spitzelii is an obligate outcrosser and lacks an effective isolating mechanism against the abundant and concurrently blooming Orchis mascula. It is therefore proposed that O. spitzelii has spread to Gotland in recent time.     

Levels of selenium in plasma and glutathione peroxidase in erythrocytes and the risk of breast cancer

Plasma selenium and glutathione peroxidase in erythrocytes were analyzed in a case-control study encompassing 441 cases with breast cancer and 191 controls with benign breast disease. No difference in mean serum selenium level between cases and controls on supplementary selenium intake was seen. If only individuals without supplementary intake, 278 cases and 135 controls, were considered a preventive effect was found increasing with selenium level. This finding was significant among women 50 years old or more with Mantel-Haenszel odds ratio=0.16 for individuals with serum selenium >1.21 μmol/L. Also for subjects with serum selenium in the range 1.00–1.21 μmol/L a significant preventive effect was seen with odds ratio=0.38. For women under 50 years of age a nonsignificant preventive effect was seen. Glutathione peroxidase in erythrocytes did not correlate well with serum selenium and was not a marker for the risk of breast cancer.     

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin-dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age-dependent constitutive activation of glycogen synthase kinase 3β, protein kinase B, mitogen-activated protein kinase, and c- Jun -N-terminal kinase in adult p62^−/− mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62^−/− brain led to recovery of aggregated K63-ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain-derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer-like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid-beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.     

Effect of pH on the association behavior in aqueous solutions of pig gastric mucin

In this study, dynamic light scattering (DLS), turbidity, and rheo-small angle light scattering (rheo-SALS) methods have been utilized to examine the impact of pH (1 < or = pH < or = 7) on aqueous solutions of noncommercial purified pig gastric mucin. The asymmetric flow field-flow fractionation (AFFFF) measurements established that the mucin sample has a high molecular weight and is polydisperse. DLS measurements on dilute solutions of mucin disclosed large interchain aggregates at pH 2, where the polymer has a low charge density or is uncharged. At lower or higher values of pH, mucin is charged and the tendency of forming interpolymer complexes is affected. In the semidilute concentration regime, pronounced junction zones ('lumps' of polymer) are evolved and a heterogeneous connected network is formed at pH 2, whereas the association structures are disintegrated (smaller 'lumps') at lower or higher pH values due to electrostatic repulsive interactions, and a more homogeneous network is evolved. The DLS and viscosity results at pH 1 indicate the development of a fragmented network, composed of contracted chains that are decorated by some positive charges. The effect of shear flow on the structure of semidilute solutions of mucin was investigated with the aid of rheo-SALS methods. The scattered intensity revealed a strong upturn at low values of the wave vector (q) for mucin solutions at pH 2 and pH 4, which suggests the evolution of large association domains. At these pH values, a flow-induced anisotropy in the 2D SALS patterns in the form of elliptical shapes was observed at high shear rates.     

Production of a truncated soluble human semicarbazide-sensitive amine oxidase mediated by a GST-fusion protein secreted from HEK293 cells

Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inflammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inflammation. To facilitate characterization and development of specific and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione S-transferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was purified in a single step by glutathione-affinity chromatography followed by site-specific cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-affinity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.