ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Introduction: Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. Methods: We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. Results: The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. Conclusions: The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.

Novel expression of type 1 corticotropin-releasing hormone receptor in multiple endocrine cell types in the murine anterior pituitary.

The CRH family of ligands signals via two distinct receptors, CRH-R1 and CRH-R2. Previous studies localized CRH-R1 and CRH-R2 to a subset of anterior pituitary corticotropes and gonadotropes, respectively. However, numerous studies have indicated that stress and CRH activity can alter the secretion of multiple anterior pituitary hormones, suggesting a broader expression of the CRH receptors in pituitary. To examine this hypothesis, the in vivo expression of CRH-R1 and CRH-R2 mRNA was further characterized in adult mouse pituitary. Quantitative RT-PCR analysis demonstrated that CRH-R1 mRNA is greater than 100-fold more abundant than CRH-R2 mRNA in male and female mouse pituitaries. Dual in situ hybridization analysis identified cell-specific CRH-R1 expression in the anterior pituitary. At least half of the CRH-R1-positive cells expressed proopiomelanocortin-mRNA (50% in females; 70% in males). In females, a significant percentage of the cells expressing CRH-R1 also expressed transcript for prolactin (40%), LHbeta (10%), or TSH (3%), all novel sites of CRH-R1 expression. Similarly in males, a percentage of CRH-R1-positive cells expressed prolactin (12%), LHbeta (13%), and TSH (5%). RT-PCR studies with immortalized murine anterior pituitary cell lines showed CRH-R1 and/or CRH-R2 expression in corticotropes (AtT-20 cells), gonadotropes (alphaT3-1 and LbetaT2 cells), and thyrotropes (alphaTSH cells). Whereas CRH-R1 expression in corticotropes is well established, the presence of CRH-R1 mRNA in a subset of lactotropes, gonadotropes, and thyrotropes establishes these cell types as novel sites of murine CRH-R1 expression and highlights the pituitary as an important site of interaction between the hypothalamus-pituitary-adrenal and multiple endocrine axes.

Effects of diet and larval trematode parasitism on lutein and beta-carotene concentrations in planorbid snails as determined by quantitative high performance reversed phase thin layer chromatography.

High performance thin layer chromatography (HPTLC) was used to quantify the concentrations of beta-carotene and lutein in Biomphalaria glabrata and Helisoma trivolvis (Colorado and Pennsylvania strains) snails under various conditions. These conditions were: snails fed a lettuce (L) vs. a yolk (Y) diet; B. glabrata infected with Echinostoma caproni vs. uninfected snails; and H. trivolvis (PA) infected with Echinostoma trivolvis vs. uninfected snails. The pigments were extracted from the snail whole bodies and digestive gland-gonad complexes, separated by reversed phase HPTLC, and quantified by densitometric scanning with standard calibration curves. Snails on the L-diet showed significant increases (Student's t-test, P<0.05) in the concentrations of beta-carotene and lutein compared to snails on the Y-diet. Snails infected with echinostomes showed no significant differences (Student's t-test, P>0.05) in the concentrations of lutein and beta-carotene compared to the uninfected cohorts. Our results were compared with previous studies that analyzed beta-carotene and lutein in snails infected with larval trematodes. Variations in the results of our study compared with others reflect intrinsic differences in the larval trematode-snail systems used.

Soluble corticotropin-releasing hormone receptor 2alpha splice variant is efficiently translated but not trafficked for secretion.

CRH directs the physiological and behavioral responses to stress. Its activity is mediated by CRH receptors (CRH-R) 1 and 2 and modulated by the CRH-binding protein. Aberrant regulation of this system has been associated with anxiety disorders and major depression, demonstrating the importance of understanding the regulation of CRH activity. An mRNA splice variant of CRH-R2alpha (sCRH-R2alpha) was recently identified that encodes the receptor's ligand-binding extracellular domain but terminates before the transmembrane domains. It was therefore predicted to serve as a secreted decoy receptor, mimicking the ability of CRH-binding protein to sequester free CRH. Although the splice variant contains a premature termination codon, predicting its degradation by nonsense-mediated RNA decay, cycloheximide experiments and polysome profiles demonstrated that sCRH-R2alpha mRNA escaped this regulation and was efficiently translated. However, the resulting protein was unable to serve as a decoy receptor because it failed to traffic for secretion because of an ineffective signal peptide and was ultimately subjected to proteosomal degradation. Several other truncated splice variants of G protein-coupled transmembrane receptors regulate the amount of full-length receptor expression through dimerization and misrouting; however, receptor binding assays and immunofluorescence of cells cotransfected with sCRH-R2alpha and CRH-R2alpha or CRH-R1 indicated that sCRH-R2alpha protein does not alter trafficking or binding of full-length CRH-R. Although sCRH-R2alpha protein does not appear to function as an intracellular or extracellular decoy receptor, the regulated unproductive splicing of CRH-R2alpha pre-mRNA to sCRH-R2alpha may selectively alter the cellular levels of full-length CRH-R2alpha mRNA and hence functional CRH-R2alpha receptor levels.