A Prospective Validation Study of Lung Cancer Gene Panel Testing Using Cytological Specimens.

Background: Genetic panel tests require sufficient tissue samples, and therefore, cannot always be performed. Although collecting cytological specimens is easier than tissue collection, there are no validation studies on the diagnostic accuracy of lung cancer gene panel tests using cytology samples. Methods: Using an amplicon-based high-sensitivity next-generation sequencing panel test capable of measuring eight druggable genes, we prospectively enrolled consecutive patients who underwent diagnostic procedures. We evaluated the analysis accuracy rate, nucleic acid yield, and the quality of cytological specimens under brushing, needle aspiration, and pleural effusion. We then compared these specimens with collected tissue samples. Results: In 163 prospectively enrolled cases, nucleic acid extraction and analysis accuracy was 100% in cases diagnosed with adenocarcinoma. Gene mutations were found in 68.7% of cases with 99.5% (95% CI: 98.2-99.9) concordance to companion diagnostics. The median DNA/RNA yield and DNA/RNA integrity number were 475/321 ng and 7.9/5.7, respectively. The correlation coefficient of the gene allele ratio in 64 cases compared with tissue samples was 0.711. Conclusion: The success of gene analysis using cytological specimens was high, and the yield and quality of the extracted nucleic acid were sufficient for panel analysis. Moreover, the allele frequency of gene mutations in cytological specimens showed high correlations with tissue specimens.

EML4-ALK fusion variant.3 and co-occurrent PIK3CA E542K mutation exhibiting primary resistance to three generations of ALK inhibitors.

The ALK inhibitors are promising therapeutic agents against lung cancer harboring ALK fusion genes and are currently under development up to the third generation. However, its therapeutic effects are reported to be affected by differences in ALK variants and co-occurrent mutations. Materials and Methods; We experienced an autopsy case of an ALK-positive lung cancer patient who showed primary resistance to three generations of ALK inhibitors. The poor survival time of the case was 14 months. To reveal the mechanism of primary resistance to three generations of ALK inhibitors, we performed next generation sequencing for 12 specimes obtained from an autopsy with covering whole exons of 53 significantly mutated, lung cancer-associated genes and amplicon-based target RNA sequenceing for the ALK fusion gene. The NGS analysis revealed a rare variant.3 of ALK fusion, in which 30 bp of base was inserted at the end of ALK intron.19 and was associated with EML exon.6 [E6_ins30A20] and a co-occurrent oncogenic PIK3CA E542K mutation in all specimens. Structural analysis of the fusion protein ALK [E6_ins30A20] showed no interferance with the binding of ALK inhibitors to the kinase domain. The NGS analysis of primary and metastatic lesions obtained from an autopsy revealed a co-occurrent oncogenic PIK3CA E542K mutation in all specimens. The constitutive activation of PI3K-Akt signal by PIK3CA E542K mutation occurred downstream of ALK signaling pathway, could lead to primary resistance to ALK inhibitors in all generations.

Practical determination of LODP (limit of detection for microarray platform) for the evaluation of microarray platforms.

The performance indicator called limit of detection for microarray platform (LODP) was defined in ISO 16578:2013. The methods to determine practical LODP were explored. In general, + 3 SD of the background is used as the signal strength of limit of detection and criteria for dividing positive and negative results. Since the negative signal had been defined differently for each microarray platform, signals obtained from Non-Probe Spots (NPS) installed on the microarrays were defined as the "background" of microarrays. LODP was determined as the lowest concentration of which the average signal exceeded Avg. + 3 SD of the background (NPS) and the signal was significantly different from those of the lower and higher adjacent concentration points measured with a diluted series of reference materials. For reliable qualitative analysis, the positive results can be defined as signals higher than those corresponding to LODP and negative results as lower signals, without determining limit of detection for all target probes. The use of LODP also enables comparisons of platform performances without checking sequence dependencies, and assists to select reliable and fitting platforms for experimental purposes.

Gastric adenocarcinoma of fundic gland mucosa type localized in the submucosa: A case report.

RATIONALE: Gastric adenocarcinoma of fundic gland type (GA-FG) is a new histological type of gastric cancer manifesting with differentiation into a fundic gland. Furthermore, gastric adenocarcinoma of fundic gland mucosa type (GA-FGM) is a tumor that shows differentiation into not only a fundic gland but also foveolar epithelium and a mucous gland. These tumors tend to invade the submucosal layer. However, no cases of these tumors being localized only in the submucosa have been reported. Here, we present a case of GA-FGM localized in the submucosa and describe the cytological features of this tumor. To our knowledge, this is the first reported case of GA-FGM localized in the submucosa. PATIENT CONCERNS: A man in his early 70s was referred to our institution because of the detection of a gastric submucosal tumor during a health checkup. DIAGNOSES: Gastric adenocarcinoma of fundic gland mucosa type. INTERVENTIONS: Endoscopic ultrasound-guided fine-needle aspiration (FNA), endoscopic submucosal dissection (ESD), and total gastrectomy with lymph node dissection were performed. OUTCOMES: The FNA specimen showed epithelial cells with low-grade atypia. In the ESD specimen, adenocarcinoma showing a gastric fundic gland mucosa-like morphology was observed. Immunohistochemical analysis showed positive staining for pepsinogen I, H+/K+-adenosine triphosphatase, MUC6, and MUC5AC and negative staining for MUC2 and CD10, indicating tumor differentiation into fundic gland mucosa. Therefore, the tumor was diagnosed as GA-FGM, with localization in the submucosal layer. Total gastrectomy and lymph node dissection were performed because of the positive margins of the ESD specimen. Neither residual tumor nor lymph node metastasis was detected; however, many foci of heterotopic gastric glands (HGGs) were observed in the gastric wall, suggesting that GA-FGM arose from an HGG. After treatment, no recurrence was observed during a 1-year follow-up period. LESSONS: Various tumors may arise from HGGs. Furthermore, when an FNA specimen shows gastric fundic gland mucosa-like epithelial cells with weak atypia, the possibility of GA-FG and GA-FGM should be considered.

FGF23 modulates the effects of erythropoietin on gene expression in renal epithelial cells.

BACKGROUND: FGF23 plays an important role in calcium-phosphorus metabolism. Other roles of FGF23 have recently been reported, such as commitment to myocardium enlargement and immunological roles in the spleen. In this study, we aimed to identify the roles of FGF23 in the kidneys other than calcium-phosphorus metabolism. METHODS: DNA microarrays and bioinformatics tools were used to analyze gene expression in mIMCD3 mouse renal tubule cells following treatment with FGF23, erythropoietin and/or an inhibitor of ERK. RESULTS: Three protein-coding genes were upregulated and 12 were downregulated in response to FGF23. Following bioinformatics analysis of these genes, PPARγ and STAT3 were identified as candidate transcript factors for mediating their upregulation, and STAT1 as a candidate for mediating their downregulation. Because STAT1 and STAT3 also mediate erythropoietin signaling, we investigated whether FGF23 and erythropoietin might show interactive effects in these cells. Of the 15 genes regulated by FGF23, 11 were upregulated by erythropoietin; 10 of these were downregulated following cotreatment with FGF23. Inhibition of ERK, an intracellular mediator of FGF23, reversed the effects of FGF23. However, FGF23 did not influence STAT1 phosphorylation, suggesting that it impinges on erythropoietin signaling through other mechanisms. CONCLUSION: Our results suggest cross talk between erythropoietin and FGF23 signaling in the regulation of renal epithelial cells.

Effects of trimethylamine N-oxide and urea on DNA duplex and G-quadruplex.

We systematically investigated effects of molecular crowding with trimethylamine N-oxide (TMAO) as a zwitterionic and protective osmolyte and urea as a nonionic denaturing osmolyte on conformation and thermodynamics of the canonical DNA duplex and the non-canonical DNA G-quadruplex. It was found that TMAO and urea stabilized and destabilized, respectively, the G-quadruplex. On the other hand, these osmolytes generally destabilize the duplex; however, it was observed that osmolytes having the trimethylamine group stabilized the duplex at the lower concentrations because of a direct binding to a groove of the duplex. These results are useful not only to predict DNA structures and their thermodynamics under physiological environments in living cells, but also design of polymers and materials to regulate structure and stability of DNA sequences.

A new class of small molecule estrogen receptor-alpha antagonists that overcome anti-estrogen resistance.

Previous studies indicate that BRCA1 protein binds to estrogen receptor-alpha (ER) and inhibits its activity. Here, we found that BRCA1 over-expression not only inhibits ER activity in anti-estrogen-resistant LCC9 cells but also partially restores their sensitivity to Tamoxifen. To simulate the mechanism of BRCA1 inhibition of ER in the setting of Tamoxifen resistance, we created a three-dimensional model of a BRCA1-binding cavity within the ER/Tamoxifen complex; and we screened a pharmacophore database to identify small molecules that could fit into this cavity. Among the top 40 "hits", six exhibited potent ER inhibitory activity in anti-estrogen-sensitive MCF-7 cells and four of the six exhibited similar activity (IC50 ≤ 1.0 µM) in LCC9 cells. We validated the model by mutation analysis. Two representative compounds (4631-P/1 and 35466-L/1) inhibited ER-dependent cell proliferation in Tamoxifen-resistant cells (LCC9 and LCC2) and partially restored sensitivity to Tamoxifen. The compounds also disrupted the association of BRCA1 with ER. In electrophoretic mobility shift assays, the compounds caused dissociation of ER from a model estrogen response element. Finally, a modified form of compound 35446 (hydrochloride salt) inhibited growth of LCC9 tumor xenografts at non-toxic concentrations. These results identify a novel group of small molecules that can overcome Tamoxifen resistance.

Drastic stabilization of parallel DNA hybridizations by a polylysine comb-type copolymer with hydrophilic graft chain.

Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a polycation backbone and dextran side chains, poly(L-lysine)-graft-dextran (PLL-g-Dex), which has been reported to form soluble interpolyelectrolyte complexes with DNA strands. We investigated the effects of PLL-g-Dex on the conformation and thermodynamics of DNA oligonucleotides forming various secondary structures. Thermodynamic analysis of the DNA structures showed that the parallel conformations involved in both DNA duplexes and triplexes were significantly and specifically stabilized by PLL-g-Dex. On the basis of thermodynamic parameters, it was further possible to design DNA switches that undergo structural transition responding to PLL-g-Dex from an antiparallel duplex to a parallel triplex even with mismatches in the third strand hybridization. These results suggest that polycationic molecules are able to induce structural polymorphism of DNA oligonucleotides, because of the conformation-selective stabilization effects.

Transcriptome profiling of brain edemas caused by influenza infection and lipopolysaccharide treatment.

Influenza A virus-associated encephalopathy triggered by influenza virus infection often occurs in children aged five and younger in Japan. However, the mechanisms behind Influenza A virus-associated encephalopathy are not yet well understood. This study developed an Influenza A virus-associated encephalopathy-like model using mice infected with Influenza A virus and given lipopolysaccharide treatment. The results showed that the mice used in the model suffered from brain edemas nearly three times more severe, as well as having higher cytokine levels in sera compared to those of the control groups. Using gene expression profiling, cytokine-related genes were found not to be up-regulated in the brain in situ, while protein coding genes, which are known to be involved in blood-brain barrier disruption, were up-regulated. Categorizing the functional groups using gene ontology revealed the terms "ion channels," "calcium oscillation," and "membrane transporter activities." The blood-brain barrier disruption found in this Influenza A virus-associated encephalopathy model can therefore be assumed to be due to a cellular electrolyte imbalance of the neuronal tissue, in addition to a cytokine storm.

Fission yeast TOR signaling is essential for the down-regulation of a hyperactivated stress-response MAP kinase under salt stress.

TOR (target of rapamycin) signaling regulates cell growth and division in response to environmental stimuli such as the availability of nutrients and various forms of stress. The vegetative growth of fission yeast cells, unlike other eukaryotic cells, is not inhibited by treatment with rapamycin. We found that certain mutations including pmc1Δ (Ca(2+)-ATPase), cps9-193 (small GTPase, Ryh1) and cps1-12 (1,3-ß-D-glucan synthase, Bgs1) confer a rapamycin-sensitive phenotype to cells under salt stress with potassium chloride (>0.5 M). Cytometric analysis revealed that the mutant cells were unable to enter the mitotic cell cycle when treated with the drug under salt stress. Gene cloning and overexpression experiments revealed that the sensitivity to rapamycin was suppressed by the ectopic expression of tyrosine phosphatases, Pyp1 and Pyp2, which are negative regulators of Spc1/Sty1 mitogen-activated protein kinase (MAPK). The level of tyrosine phosphorylation on Spc1 was higher and sustained substantially longer in these mutants than in the wild type under salt stress. The hyperphosphorylation was significantly suppressed by overexpression of pyp1 (+) with concomitant resumption of the mutant cells' growth. In fission yeast, TOR signaling has been thought to stimulate the stress-response pathway, because mutations of TORC2 components such as Tor1, Sin1 and Ste20 result in similar sensitive phenotypes to environmental stress. The present study, however, strongly suggests that TOR signaling is required for the down-regulation of a hyperactivated Spc1 for reentry into the mitotic cell cycle. This finding may shed light on our understanding of a new stress-responsive mechanism in TOR signaling in higher organisms.

Effect of heel lifts on plantarflexor and dorsiflexor activity during gait.

BACKGROUND: Previous investigators have shown that high heels decrease the muscle activity of the gastrocnemius muscle during gait. However, it is not known whether commonly used in-shoe heel lifts of lower heights will demonstrate similar effects on muscle activity. The aim of this study was to determine whether heel lifts alter the muscle activity of the ankle plantarflexors and dorsiflexors during the stance phase of gait among individuals with limited gastrocnemius extensibility. MATERIALS AND METHODS: This study used a repeated measures design. Twenty-four healthy volunteers (12 males and 12 females) with less than 5 degrees of passive ankle dorsiflexion with the knee extended participated in the study. Electromyography (EMG), computerized motion analysis, and a force plate were used to measure mean muscle activity of the lateral gastrocnemius, medial gastrocnemius, soleus and tibialis anterior muscles during the stance phase of gait across three walking conditions. Muscle activity was measured as participants ambulated at a self-selected speed in athletic shoes alone and with heel lifts of 6 mm and 9 mm inserted in athletic shoes. RESULTS: Between heel-strike and heel-off, the mean EMG amplitude of the medial gastrocnemius increased with both 6 and 9 mm heel lifts and the amplitude of the tibialis anterior increased with 9 mm heel lifts compared to shoes alone. Between heel-strike and heel-off, there were no significant differences in mean EMG amplitude of the lateral gastrocnemius or soleus muscles walking in heel lifts compared to shoes alone. Between heel-off and toe-off, there were no significant differences in mean EMG amplitude of the lateral gastrocnemius, medial gastrocnemius, soleus, or tibialis anterior muscles when walking in heel lifts compared to shoes alone. CONCLUSION: Heel lifts increase muscle activity of the medial gastrocnemius and tibialis anterior muscles between heel-strike and heel-off among individuals with limited gastrocnemius extensibility. CLINICAL RELEVANCE: We were unable to confirm a decrease in muscle activity when using heel lifts.

Comparative quantitative mass spectrometry analysis of MHC class II-associated peptides reveals a role of GILT in formation of self-peptide repertoire.

Gamma interferon Inducible Lysosomal Thiol reductase (GILT) is a unique lysosomal reductase that reduces disulfide bonds of endocytosed proteins. Lack of GILT clearly decreases CD4 T cell-antigen specific responses against some epitopes of antigens containing disulfide bonds, but not to proteins with few or no disulfide bridges. Hence, global impact of GILT on antigen presentation is currently not well understood. We used Nano-LC-ESI-MS/MS to investigate how GILT affects diversity of self-peptides presented by MHC class II molecules. Surprisingly, the repertoire of self-peptides in the absence of GILT does not appear to be significantly different, as only few peptide species (approximately 2%) were found to be the unique indicators of GILT's presence or absence. In the absence of GILT about thirty peptide species (approximately 5%) were found either uniquely or fourteen to hundred fold more abundantly expressed than in the presence of GILT. Our data indicate that GILT has limited yet unexpected effect on self-peptide species presented by MHC class II antigens.

Altered GABAergic neurotransmission is associated with increased kainate-induced seizure in prostaglandin-endoperoxide synthase-2 deficient mice.

Excitotoxicity involves over activation of brain excitatory glutamate receptors and has been implicated in neurological, neurodegenerative and neuropsychiatric diseases. Metabolism of arachidonic acid (AA) through the phospholipase A(2) (PLA(2))/prostaglandin-endoperoxide synthase (PTGS) pathway is increased after excitotoxic stimulation. However, the individual roles of the PTGS isoforms in this process are not well established. We assessed the role of the PTGS isoforms in the process of excitotoxicity by exposing mice deficient in either PTGS-1 (PTGS-1(-/-)) or PTGS-2 (PTGS-2(-/-)) to the prototypic excitotoxin, kainic acid (KA). Seizure intensity and neuronal damage were significantly elevated in KA-exposed PTGS-2(-/-), but not in PTGS-1(-/-), mice. The increased susceptibility was not associated with an alteration in KA receptor binding activity or mediated through the CB1 endocannabinoid receptor. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) was decreased in the CA1 pyramidal neurons of PTGS-2(-/-) mice, suggesting an alteration of GABAergic function. In wild-type mice, six weeks treatment with the PTGS-2 selective inhibitor celecoxib recapitulated the increased susceptibility to KA-induced excitotoxicity observed in PTGS-2(-/-) mice, further supporting the role of PTGS-2 in the excitotoxic process. The increased susceptibility to KA was also associated with decreased brain levels of PGE(2), a biomarker of PTGS-2 activity. Our results suggest that PTGS-2 activity and its specific products may modulate neuronal excitability by affecting GABAergic neurotransmission. Further, inhibition of PTGS-2, but not PTGS-1, may increase the susceptibility to seizures.

Design, synthesis, and characterization of a potent, nonpeptide, cell-permeable, bivalent Smac mimetic that concurrently targets both the BIR2 and BIR3 domains in XIAP.

XIAP is a central apoptosis regulator that inhibits apoptosis by binding to and inhibiting the effectors caspase-3/-7 and an initiator caspase-9 through its BIR2 and BIR3 domains, respectively. Smac protein in its dimeric form effectively antagonizes XIAP by concurrently targeting both its BIR2 and BIR3 domains. We report the design, synthesis, and characterization of a nonpeptide, cell-permeable, bivalent small-molecule (SM-164) which mimics Smac protein for targeting XIAP. Our study shows that SM-164 binds to XIAP containing both BIR domains with an IC50 value of 1.39 nM, being 300 and 7000 times more potent than its monovalent counterparts and the natural Smac AVPI peptide, respectively. SM-164 concurrently interacts with both BIR domains in XIAP and functions as an ultrapotent antagonist of XIAP in both cell-free functional and cell-based assays. SM-164 targets cellular XIAP and effectively induces apoptosis at concentrations as low as 1 nM in the HL-60 leukemia cell line. The potency of bivalent SM-164 in binding, functional, and cellular assays is 2-3 orders of magnitude higher than its corresponding monovalent Smac mimetics.

Identification of the fibroblast growth factor (FGF)-interacting domain in a secreted FGF-binding protein by phage display.

Fibroblast growth factor-binding proteins (FGF-BP) are secreted carrier proteins that release fibroblast growth factors (FGFs) from the extracellular matrix storage and thus enhance FGF activity. Here we have mapped the interaction domain between human FGF-BP1 and FGF-2. For this, we generated T7 phage display libraries of N-terminally and C-terminally truncated FGF-BP1 fragments that were then panned against immobilized FGF-2. From this panning, a C-terminal fragment of FGF-BP1 (amino acids 193-234) was identified as the minimum binding domain for FGF. As a recombinant protein, this C-terminal fragment binds to FGF-2 and enhances FGF-2-induced signaling in NIH-3T3 fibroblasts and GM7373 endothelial cells, as well as mitogenesis and chemotaxis of NIH-3T3 cells. The FGF interaction domain in FGF-BP1 is distinct from the heparin-binding domain (amino acids 110-143), and homology modeling supports the notion of a distinct domain in the C terminus that is conserved across different species. This domain also contains conserved positioning of cysteine residues with the Cys-214/Cys-222 positions in the human protein predicted to participate in disulfide bridge formation. Phage display of a C214A mutation of FGF-BP1 reduced binding to FGF-2, indicating the functional significance of this disulfide bond. We concluded that the FGF interaction domain is contained in the C terminus of FGF-BP1.