Andreas S. Beutler

Somatic mutation of CDKN1B in small intestine neuroendocrine tumors

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not yet been defined. Using exome- and 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-NETs.

The genomic landscape of small intestine neuroendocrine tumors

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 106 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.

mRNA-seq with agnostic splice site discovery for nervous system transcriptomics tested in chronic pain.

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.

Clinical Course of Oxaliplatin-Induced Neuropathy: Results From the Randomized Phase III Trial N08CB (Alliance)

PURPOSE Given that the clinical course of oxaliplatin-induced neuropathy is not well defined, the current study was performed to better understand clinical parameters associated with its presentation. METHODS Acute and chronic neuropathy was evaluated in patients receiving adjuvant FOLFOX (fluorouracil, leucovorin, and oxaliplatin) on study N08CB (North Central Cancer Treatment Group, Alliance). Acute neuropathy was assessed by having patients complete daily questionnaires for 6 days with each cycle of FOLFOX. Before each dose of FOLFOX and as long as 18 months after chemotherapy cessation, chronic neurotoxicity was assessed with use of the 20-item, European Organisation for Research and Treatment of Cancer quality-of-life questionnaire for patients with chemotherapy-induced peripheral neuropathy. RESULTS Three hundred eight (89%) of the 346 patients had at least one symptom of acute neuropathy with the first cycle of FOLFOX; these symptoms included sensitivity to touching cold items (71%), sensitivity to swallowing cold items (71%), throat discomfort (63%), or muscle cramps (42%). Acute symptoms peaked at day 3 and improved, although they did not always resolve completely between treatments. These symptoms were about twice as severe in cycles 2 through 12 as they were in cycle 1. For chronic neurotoxicity, tingling was the most severe symptom, followed by numbness and then pain. During chemotherapy, symptoms in the hands were more prominent than they were in the feet; by 18 months, symptoms were more severe in the feet than they were in the hands. Patients with more severe acute neuropathy during the first cycle of therapy experienced more chronic sensory neurotoxicity (P < .0001). CONCLUSION 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.

Sequencing of Charcot-Marie-Tooth disease genes in a toxic polyneuropathy.

Mutations in Charcot–Marie–Tooth disease (CMT) genes are the cause of rare familial forms of polyneuropathy. Whether allelic variability in CMT genes is also associated with common forms of polyneuropathy—considered “acquired” in medical parlance—is unknown. Chemotherapy‐induced peripheral neuropathy (CIPN) occurs commonly in cancer patients and is individually unpredictable. We used CIPN as a clinical model to investigate the association of non‐CMT polyneuropathy with CMT genes.

SAAP-RRBS

Summary: Reduced representation bisulfite sequencing (RRBS) is a cost-effective approach for genome-wide methylation pattern profiling. Analyzing RRBS sequencing data is challenging and specialized alignment/mapping programs are needed. Although such programs have been developed, a comprehensive solution that provides researchers with good quality and analyzable data is still lacking. To address this need, we have developed a Streamlined Analysis and Annotation Pipeline for RRBS data (SAAP-RRBS) that integrates read quality assessment/clean-up, alignment, methylation data extraction, annotation, reporting and visualization. This package facilitates a rapid transition from sequencing reads to a fully annotated CpG methylation report to biological interpretation. Availability and implementation: SAAP-RRBS is freely available to non-commercial users at the web site http://ndc.mayo.edu/mayo/research/biostat/stand-alone-packages.cfm. Contact: baheti.saurabh@mayo.edu or sun.zhifu@mayo.edu Supplementary Information: Supplementary data are available at Bioinformatics online.

Exome-level comparison of primary well-differentiated neuroendocrine tumors and their cell lines

Neuroendocrine cancer cell lines are used to investigate therapeutic targets in neuroendocrine tumors (NET) and have been instrumental in the design of clinical trials targeting the PI3K/AKT/mTOR pathways, VEGF inhibitors, and somatostatin analogues. It remains unknown, however, whether the genomic makeup of NET cell lines reflect that of primary NET since comprehensive unbiased genome sequencing has not been performed on the cell lines. Four bronchopulmonary NET (BP-NET)-NCI-H720, NCI-H727, NCI-H835, and UMC11-and two pancreatic neuroendocrine tumors (panNET)-BON-1 and QGP1-were cultured. DNA was isolated, and exome sequencing was done. GATK and EXCAVATOR were used for bioinformatic analysis. We detected a total of 1,764 nonsynonymous single nucleotide variants at a rate of 8 per Mb in BP-NET and 4.3 per Mb in panNET cell lines, including 52 mutated COSMIC cancer genes in these cell lines, such as TP53, BRCA1, RB1, TSC2, NOTCH1, EP300, GNAS, KDR, STK11, and APC but not ATRX, DAXX, nor MEN1. Our data suggest that mutation rate, the pattern of copy number variations, and the mutational spectra in the BP-NET cell lines are more similar to the changes observed in small cell lung cancer than those found in primary BP-NET. Likewise, mutation rate and pattern including the absence of mutations in ATRX/DAXX, MEN1, and YY1 in the panNET cell lines BON1 and QGP1 suggest that these cell lines do not have the genetic signatures of a primary panNET. These results suggest that results from experiments with BP-NET and panNET cell lines need to be interpreted with caution.

SERE: Single-parameter quality control and sample comparison for RNA-Seq

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.

Diametrically opposite methylome-transcriptome relationships in high- and low-CpG promoter genes in postmitotic neural rat tissue.

DNA methylation can control some CpG-poor genes but unbiased studies have not found a consistent genome-wide association with gene activity outside of CpG islands or shores possibly due to use of cell lines or limited bioinformatics analyses. We performed reduced representation bisulfite sequencing (RRBS) of rat dorsal root ganglia encompassing postmitotic primary sensory neurons (n = 5, r > 0.99; orthogonal validation p < 10−19). The rat genome suggested a dichotomy of genes previously reported in other mammals: low CpG content (< 3.2%) promoter (LCP) genes and high CpG content (≥ 3.2%) promoter (HCP) genes. A genome-wide integrated methylome-transcriptome analysis showed that LCP genes were markedly hypermethylated when repressed, and hypomethylated when active with a 40% difference in a broad region at the 5′ of the transcription start site (p < 10−87 for -6000 bp to -2000 bp, p < 10−73 for -2000 bp to +2000 bp, no difference in gene body p = 0.42). HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes. Therefore, diametrically opposite methylome-transcriptome associations characterize LCP and HCP genes in postmitotic neural tissue in vivo.

National Cancer Institute-supported chemotherapy-induced peripheral neuropathy trials: outcomes and lessons

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.