Feasibility and clinical utility of endoscopic ultrasound guided biopsy of pancreatic cancer for next-generation molecular profiling.

Next-generation sequencing is enabling molecularly guided therapy for many cancer types, yet failure rates remain relatively high in pancreatic cancer (PC). The aim of this study is to investigate the feasibility of genomic profiling using endoscopic ultrasound (EUS) biopsy samples to facilitate personalised therapy for PC. Ninty-five patients underwent additional research biopsies at the time of diagnostic EUS. Diagnostic formalin-fixed (FFPE) and fresh frozen EUS samples underwent DNA extraction, quantification and targeted gene sequencing. Whole genome (WGS) and RNA sequencing was performed as proof of concept. Only 2 patients (2%) with a diagnosis of PC had insufficient material for targeted sequencing in both FFPE and frozen specimens. Targeted panel sequencing (n=54) revealed mutations in PC genes (KRAS, GNAS, TP53, CDKN2A, SMAD4) in patients with histological evidence of PC, including potentially actionable mutations (BRCA1, BRCA2, ATM, BRAF). WGS (n=5) of EUS samples revealed mutational signatures that are potential biomarkers of therapeutic responsiveness. RNA sequencing (n=35) segregated patients into clinically relevant molecular subtypes based on transcriptome. Integrated multi-omic analysis of PC using standard EUS guided biopsies offers clinical utility to guide personalized therapy and study the molecular pathology in all patients with PC

Nitric oxide metabolite levels and assessment of cervical length in the prediction of preterm delivery among women undergoing symptomatic preterm labor

Objective: To evaluate the diagnostic accuracy of measuring cervical length (CL) in combination with cervical and plasma nitric oxide metabolite (NOx) levels to identify women undergoing preterm labor (PTL) who will deliver preterm. Methods: A hospital-based prospective cohort study of 730 women undergoing spontaneous PTL between 24 and 33 weeks + 6 days of pregnancy was conducted. Measurement of cervical and plasma NOx levels and ultrasonographic assessment of CL were performed to find the best model to predict preterm delivery (PTD). Optimal cut-off values were calculated by receiver operating characteristic (ROC) curve analysis. Logistic regression analysis and rank correlation tests were also performed. Results: CL of 15 mm or less, cervical NOx levels greater than 87.6 mu mol/L, and plasma NOx levels greater than 123 mu mol/L (P<0.0001) were the only factors significantly associated with PTD within 7 days of sampling. This combined model provided high diagnostic accuracy (sensitivity 80.0%; specificity 99.2%). Both cervical and plasma NOx levels were negatively correlated with CL (r=-0.453, P<0.0001 and r=-0.362, P<0.0001, respectively). Conclusion: Combined measurement of CL and levels of cervical and plasma NOx could help identify women undergoing symptomatic PTL who are at increased risk of PTD. (C) 2011 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved

Generation of biologically active retro-genes upon interaction of mouse spermatozoa with exogenous DNA

Mature spermatozoa of most animal species can spontaneously take up foreign DNA molecules which can be delivered to embryos upon fertilization. Following this procedure, transgenic animals of various species have been generated. We recently discovered a reverse transcriptase (RT) activity in mouse spermatozoa that can reverse-transcribe exogenous RNA molecules into cDNA copies. These cDNA copies are transferred to embryos at fertilization, mosaic propagated as non-integrated structures in tissues of founder individuals and further transmitted to F1 progeny. Reverse-transcribed sequences behave as functional genes, being correctly expressed in tissues of F0 and F1 animals. To learn more about this mechanism and further characterize the reverse transcription step, we have now incubated spermatozoa with a plasmid harboring a green fluorescent protein (EGFP) retrotransposition cassette interrupted by an intron in the opposite orientation to the EGFP gene. We found that reverse-transcribed spliced EGFP DNA sequences are generated in sperm cells and transmitted to embryos in IVF assays. After implantation in foster mothers, embryos developed into mice that expressed EGFP in the blood vessel endothelia of a variety of organs. The EGFP-encoding cDNA sequences were detected in positive tissues as extrachromosomal mosaic-propagated structures, maintained in low-copy number (< 1 copy/genome), and mosaic transmitted from founders to the F1 progeny. These results indicate that an efficient machinery is present in mature spermatozoa, which can transcribe, splice, and reverse-transcribe exogenous DNA molecules. This mechanism is implicated in the genesis and non-Mendelian propagation of new genetic information besides that contained in chromosomes

A tailored mouse model of CLN2 disease: A nonsense mutant for testing personalized therapies

<div><p>The Neuronal Ceroid Lipofuscinoses (NCLs), also known as Batten disease, result from mutations in over a dozen genes. Although, adults are susceptible, the NCLs are frequently classified as pediatric neurodegenerative diseases due to their greater pediatric prevalence. Initial clinical presentation usually consists of either seizures or retinopathy but develops to encompass both in conjunction with declining motor and cognitive function. The NCLs result in premature death due to the absence of curative therapies. Nevertheless, preclinical and clinical trials exist for various therapies. However, the genotypes of NCL animal models determine which therapeutic approaches can be assessed. Mutations of the <i>CLN2</i> gene encoding a soluble lysosomal enzyme, tripeptidyl peptidase 1 (TPP1), cause late infantile NCL/CLN2 disease. The genotype of the original mouse model of CLN2 disease, <i>Cln2</i>^<i>-/-</i>, excludes mutation guided therapies like antisense oligonucleotides and nonsense suppression. Therefore, the purpose of this study was to develop a model of CLN2 disease that allows for the assessment of all therapeutic approaches. Nonsense mutations in CLN2 disease are frequent, the most common being <i>CLN2</i>^<i>R208X</i>. Thus, we created a mouse model that carries a mutation equivalent to the human p.R208X mutation. Molecular assessment of <i>Cln2</i>^{<i>R207X/R207X</i>} tissues determined significant reduction in <i>Cln2</i> transcript abundance and TPP1 enzyme activity. This reduction leads to the development of neurological impairment (e.g. tremors) and neuropathology (e.g. astrocytosis). Collectively, these assessments indicate that the <i>Cln2</i>^{<i>R207X/R207X</i>} mouse is a valid CLN2 disease model which can be used for the preclinical evaluation of all therapeutic approaches including mutation guided therapies.</p></div

Lysosomal accumulation of mitochondrial ATP synthase subunit c in <i>Cln2</i>^{<i>R207X/R207X</i>} mice.

<p>Subunit c accumulation was detected by immunofluorescent staining. (A) Images of superficial and deep cortical layers demonstrate diffuse and pronounced accumulation of mitochondrial ATP synthase subunit c in 3-month-old <i>Cln2</i>^{<i>R207X/R207X</i>} mice. (B) Images from 3-month-old <i>Cln2</i>^<i>+/+</i> (n = 4) and <i>Cln2</i>^{<i>R207X/R207X</i>} (n = 5) mice were blindly collected and analyzed for differences in cell number, total number of immunoreactive puncta, number of puncta per cell, and average punctum area. <i>Cln2</i>^{<i>R207X/R207X</i>} mice have significantly increased total number of puncta, puncta per cell, and punctum size. Columns and bars represent mean ± SEM. Statistical significance was determined using an unpaired t-test (**p <u><</u> 0.01, ***p <u><</u> 0.001, and **** p < 0.0001).</p

Frequency of CLN2 disease-associated mutations.

<p>(A) Allele frequency of CLN2 disease mutations demonstrates a predominance of nonsense (29%) and missense (22%) mutations. (B) The most common CLN2 disease mutations consists of either the intronic transversion c.509-1G>C that results in altered transcript splicing or the exonic transition c.622C>T that results in the p.R208X nonsense mutation.</p