The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not been defined for these tumors. Using exome/genome sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and we detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NET.

Small intestine neuroendocrine tumors (SI-NETs) are the most common malignancy of the small bowel. Several clinical trials target PI3K/Akt/mTOR signaling; however, it is unknown whether these or other genes are genetically altered in these tumors. To address the underlying genetics, we analyzed 48 SI-NETs by massively parallel exome sequencing. We detected an average of 0.1 somatic single nucleotide variants (SNVs) per 10 6 nucleotides (range, 0-0.59), mostly transitions (C>T and A>G), which suggests that SI-NETs are stable cancers. 197 protein-altering somatic SNVs affected a preponderance of cancer genes, including FGFR2, MEN1, HOOK3, EZH2, MLF1, CARD11, VHL, NONO, and SMAD1. Integrative analysis of SNVs and somatic copy number variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis, RAS signaling, and axon guidance. Candidate therapeutically relevant alterations were found in 35 patients, including SRC, SMAD family genes, AURKA, EGFR, HSP90, and PDGFR. Mutually exclusive amplification of AKT1 or AKT2 was the most common event in the 16 patients with alterations of PI3K/Akt/ mTOR signaling. We conclude that sequencing-based analysis may provide provisional grouping of SI-NETs by therapeutic targets or deregulated pathways. IntroductionSmall intestine neuroendocrine neoplasms (SI-NENs) are the most common malignancy of the small bowel, represent the largest group of NENs by organ site, and are studied in clinical treatment trials targeting PI3K/Akt/mTOR signaling. Whether this or other canonical cancer pathways is recurrently mutated, however, is uncertain, because a genome-wide, unbiased sequence analysis of cancer genes has not been performed to date in SI-NENs.Massively parallel, or "nextgen," DNA sequencing is currently advancing research in other human malignancies by facilitating the collection of comprehensive, genome-wide, unbiased datasets providing a common data framework for comparing results across different tumor types and gene sets. It provides the most comprehensive technology to date to explore the potential of genomics for individualizing cancer treatment within a tumor type. To unlock and explore the potential of the technology for translational research in SI-NEN, we sequenced 48 such tumors.

Acute oxaliplatin-induced neuropathy symptoms do not always completely resolve between treatment cycles and are only half as severe on the first cycle as compared with subsequent cycles. There is a correlation between the severities of acute and chronic neuropathies.

Lumbar puncture (LP) is an attractive route to deliver drugs to the nervous system because it is a safe bedside procedure. Its use for gene therapy has been complicated by poor vector performance and failure to target neurons. Here we report highly effective gene transfer to the primary sensory neurons of the dorsal root ganglia (DRGs) with self-complementary recombinant adeno-associated virus serotype 8 (sc-rAAV8) modeling an LP. Transgene expression was selective for these neurons outlining their cell bodies in the DRGs and their axons projecting into the spinal cord. Immunohistochemical studies demonstrated transduction of cells positive for the nociceptive neuron marker vanilloid receptor subtype 1, the small peptidergic neuron markers substance P and calcitonin generelated peptide, and the nonpeptidergic neuron marker griffonia simplicifolia isolectin B4. We tested the efficacy of the approach in a rat model of chronic neuropathic pain. A single administration of sc-rAAV8 expressing the analgesic gene prepro-␤-endorphin (pp␤EP) led to significant (P < 0.0001) reversal of mechanical allodynia for >3 months. The antiallodynic effect could be reversed by the -opioid antagonist naloxone 4 months after gene transfer (P < 0.001). Testing of an alternative nonopioid analgesic gene, IL-10, alone or in combination with pp␤EP was equally effective (P < 0.0001). All aspects of the procedure, such as the use of an atraumatic injection technique, isotonic diluent, a low-infusion pressure, and a small injection volume, are consistent with clinical practice of intrathecal drug use. Therefore, gene transfer by LP may be suitable for developing gene therapy-based treatments for chronic pain.adeno-associated virus ͉ dorsal root ganglion ͉ gene therapy ͉ ␤-endorphin ͉ IL-10

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BackgroundAssessing the reliability of experimental replicates (or global alterations corresponding to different experimental conditions) is a critical step in analyzing RNA-Seq data. Pearson’s correlation coefficient r has been widely used in the RNA-Seq field even though its statistical characteristics may be poorly suited to the task.ResultsHere we present a single-parameter test procedure for count data, the Simple Error Ratio Estimate (SERE), that can determine whether two RNA-Seq libraries are faithful replicates or globally different. Benchmarking shows that the interpretation of SERE is unambiguous regardless of the total read count or the range of expression differences among bins (exons or genes), a score of 1 indicating faithful replication (i.e., samples are affected only by Poisson variation of individual counts), a score of 0 indicating data duplication, and scores >1 corresponding to true global differences between RNA-Seq libraries. On the contrary the interpretation of Pearson’s r is generally ambiguous and highly dependent on sequencing depth and the range of expression levels inherent to the sample (difference between lowest and highest bin count). Cohen’s simple Kappa results are also ambiguous and are highly dependent on the choice of bins. For quantifying global sample differences SERE performs similarly to a measure based on the negative binomial distribution yet is simpler to compute.ConclusionsSERE can therefore serve as a straightforward and reliable statistical procedure for the global assessment of pairs or large groups of RNA-Seq datasets by a single statistical parameter.

mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.[Supplemental material is available online at http://www.genome.org. Sequence reads from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession no. GSE20895. Software is available for download from http://www.th-wildau.de/bioinformatics/wios/ and http://mayoresearch.mayo.edu/ mayo/research/beutler_lab/wios.cfm.]Microarray-based transcriptome studies have provided a productive approach to the discovery of therapeutic targets in neurological disorders. For example, gene expression profiling of the dorsal root ganglion (DRG) in chronic pain (Griffin et al. 2003) identified several altered genes (Costigan et al. 2002), some of which were subsequently shown to be key modulators of pain (Tegeder et al. 2006). Gene expression analysis using microarray technology suffers from well-known limitations including poor sensitivity and dynamic range, a requirement for substantial requisite amounts of RNA, and a limited capacity to identify new transcripts or RNA splice sites. Given these limitations, it is unlikely that microarray analysis can reveal the full extent of transcriptome reprogramming underlying neurological disorders, such as chronic pain.Ultra-high-throughput RNA sequencing has emerged as a revolutionary technology with superior...

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the most common and debilitating complications of cancer treatment. Due to a lack of effective management options for patients with CIPN, the National Cancer Institute (NCI) sponsored a series of trials aimed at both prevention and treatment. A total of 15 such studies were approved, evaluating use of various neuro-modulatory agents which have shown benefit in other neuropathic pain states. Aside from duloxetine, none of the pharmacologic methods demonstrated therapeutic benefit for patients with CIPN. Despite these disappointing results, the series of trials revealed important lessons that have informed subsequent work. Some examples of this include the use of patient-reported symptom metrics, the elimination of traditional—yet unsubstantiated—practice approaches, and the discovery of molecular genetic predictors of neuropathy. Current inquiry is being guided by the results from these large-scale trials, and as such, stands better chance of identifying durable solutions for this treatment-limiting toxicity.