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Figure S1. Generation of the LSL-N-RasQ61R allele. Figure S2. Conditional expression of N-Ras mutants does not alter melanocyte morphology. Figure S3. In both the heterozygous and homozygous state, NRasG12D fails to induce melanoma formation. Figure S4. Melanocyte-specific N-RASQ61R expression causes high penetrance nevus formation. Figure S5. Characteristics of Ras-mutant murine melanocytes and melanomas. Figure S6. Establishment and validation of TpLN61R/61R tumor cell lines. Figure S7. Binding of NRAS61R-mant-GMPPNP to PI3Kγ and RAF-RBD. Figure S8. NRASG12D and NRASQ61R similarly bind BRAF as measured by isothermal titration calorimetry. Figure S9. Proliferation of NRAS mutant human melanoma cell lines is not codon-specific. Figure S10. Activation of ERK and AKT in melanocytes is not codon-specific. Table S1. Frequency of canonical RAS mutations in human cancer. Table S2. Comparative stability of GMPPCP- and GDP- bound NRAS variants. Table S3. Summary of prior RAS-driven murine melanoma models.

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways.Significance: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional “knock-in” mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation. Cancer Discov; 4(12); 1418–29. ©2014 AACR.This article is highlighted in the In This Issue feature, p. 1355

Machine learning has seen slow but steady uptake in diagnostic pathology over the past decade to assess digital whole slide images. Machine learning tools have incredible potential to standardize, and likely even improve, histopathologic diagnoses, but they are not yet widely used in clinical practice. We describe here the principles of these tools and technologies and some successful pre-clinical and pre-translational efforts in cardiovascular pathology, as well as a roadmap for moving forward. In animal models, one proof-of-principle application is in rodent progressive cardiomyopathy, of particular significance to drug toxicity studies. Basic science successes include screening the quality of differentiated stem cells and characterizing cardiomyocyte developmental stages, with potential applications for research and toxicology/drug safety screening using derived or native human pluripotent stem cells differentiated into cardiomyocytes. Translational studies of particular note include those with success in diagnosing the various forms of heart allograft rejection. In fully realizing the value of these tools in clinical cardiovascular pathology, we have identified three essential challenges. First is image quality standardization to ensure that algorithms can be developed and implemented on robust, consistent data. The second is consensus diagnosis; experts don't always agree, and thus "truth" may be difficult to establish, but the algorithms themselves may provide a solution. The third is the need for large enough data sets to facilitate robust algorithm development, necessitating large, cross-institutional, shared image databases. The power of histopathology-based machine learning technologies is tremendous; we outline here the next steps needed to capitalize on this power.

Summary Primary hepatic neoplasms with neuroendocrine differentiation are extremely rare. Their clinicopathological features and molecular genetic basis are largely unknown. We identified four cases of primary hepatic neoplasms with neuroendocrine differentiation. Electronic medical records were reviewed for clinical history, imaging findings, laboratory results, and follow-up. Pathology slides, immunohistochemistry, and ancillary studies were reviewed. There were two females and two males with age ranging from 52 to 74 years. There was one amphicrine carcinoma with tumor cells simultaneously demonstrating both hepatocellular and neuroendocrine differentiation, one mixed hepatocellular-neuroendocrine carcinoma (NEC) with hepatocellular component intermingled with neuroendocrine component, one small cell NEC, and one well-differentiated neuroendocrine tumor. Next- generation sequencing of the mixed hepatocellular-NEC and small cell NEC showed molecular/genetic alterations commonly seen in hepatocellular carcinoma (HCC). All four cases arose in a background of cirrhosis. Primary hepatic neoplasms arising in cirrhotic livers can have a spectrum of neuroendocrine differentiation. Presence of a NEC component may be an indicator of aggressiveness. In addition, primary hepatic carcinomas with neuroendocrine differentiation likely share the same molecular pathways as HCC.

The identification of gene fusions is a cornerstone of diagnosis and treatment decision making for tumors of many forms and primary sites. Diverse molecular approaches are currently used for the molecular diagnosis of fusions, but few permit broad, partner agnostic detection of fusions over multiple potential targets. We previously described the combination of nanopore sequencing with the anchored multiplex PCR technique to permit a rapid testing paradigm. Recently, a new platform for nanopore sequencing has become publicly available, the Flongle flow cell from Oxford Nanopore Technologies, which offers lower throughput, but lower price testing. Here, we describe the results of retesting of 15 specimens previously tested with both Illumina and Oxford Nanopore Technologies MinION sequencing. Furthermore, we additionally blindly tested 13 specimens that had undergone clinical Illumina-based sequencing. The Flongle sequencing pipeline removed key complexities of a multiplexed nanopore sequencing protocol, reduced sequencing turnaround time, and showed excellent concordance with Illumina results. It was particularly strong in identifying notoriously difficult to detect CIC-DUX4 translocations. The Flongle sequencing pipeline may be the assay of choice for deployment in small- to medium-sized molecular laboratories.

Carbon monoxide (CO) has long been considered a toxic gas but is now a recognized bioactive gasotransmitter with potent immunomodulatory effects. Although inhaled CO is currently under investigation for use in patients with lung disease, this mode of administration can present clinical challenges. The capacity to deliver CO directly and safely to the gastrointestinal (GI) tract could transform the management of diseases affecting the GI mucosa such as inflammatory bowel disease or radiation injury. To address this unmet need, inspired by molecular gastronomy techniques, we have developed a family of gas-entrapping materials (GEMs) for delivery of CO to the GI tract. We show highly tunable and potent delivery of CO, achieving clinically relevant CO concentrations in vivo in rodent and swine models. To support the potential range of applications of foam GEMs, we evaluated the system in three distinct disease models. We show that a GEM containing CO dose-dependently reduced acetaminophen-induced hepatocellular injury, dampened colitis-associated inflammation and oxidative tissue injury, and mitigated radiation-induced gut epithelial damage in rodents. Collectively, foam GEMs have potential paradigm-shifting implications for the safe therapeutic use of CO across a range of indications.

There is a critical need for sensitive and rapid detection technologies utilizing molecular biotargets such as microRNAs (miRNAs), which regulate gene expression and are a promising class of diagnostic biomarkers for disease detection. Here, we present the development and fabrication of a highly reproducible and robust plasmonic bimetallic nanostar biosensing platform to detect miRNA targets using surfaced-enhanced Raman scattering (SERS)-based gene probes called the inverse Molecular Sentinel (iMS). We investigated and optimized the integration of iMS gene probes onto this SERS substrate, achieving ultra-sensitive detection with limits of detection of 6.8 and 16.7 zmol within the sensing region for two miRNA sequences of interest. Finally, we demonstrated the biomedical usefulness of this nanobiosensor platform with the multiplexed detection of upregulated miRNA targets, miR21 and miR221, from colorectal cancer patient plasma. The resulting SERS data are in excellent agreement with PCR data obtained from patient samples and can distinguish between healthy and cancerous patient samples. These results underline the potential of the iMS-integrated substrate nanobiosensing platform for rapid and sensitive diagnostics of cancer biomarkers for point-of-care applications.

IntroductionIn colitis, macrophage functionality is altered compared to homeostatic conditions. Loss of IL-10 signaling results in an inappropriate and chronic inflammatory response to bacterial stimulation. It remains unknown if inhibition of bromodomain and extra-terminal domain (BET) proteins alters usage of DNA regulatory elements responsible for driving inflammatory gene expression. We determined if the BET inhibitor, (+)-JQ1, could suppress inflammatory activation of macrophages in Il10-/- mice.MethodsWe performed ATAC-seq and RNA-seq on Il10-/- bone marrow-derived macrophages (BMDMs) cultured in the presence or absence of lipopolysaccharide (LPS) and with or without treatment with (+)-JQ1 and evaluated changes in chromatin accessibility and gene expression. Germ-free Il10-/- mice were treated with (+)-JQ1, colonized with fecal slurries and underwent histological and molecular evaluation 14-days post colonization.ResultsTreatment with (+)-JQ1 suppressed LPS-induced changes in chromatin at distal regulatory elements associated with inflammatory genes, particularly in regions that contain motifs for AP-1 and IRF transcription factors. This resulted in the attenuation of inflammatory gene expression. Treatment with (+)-JQ1 in vivo reduced severity of colitis as compared with vehicle-treated mice.ConclusionWe identified the mechanism of action associated with a new class of compounds that may mitigate aberrant macrophage responses to bacteria in colitis.FundingThis work was funded in part through Helmsley Charitable Trust (SHARE Project 2), NIDDK P01DK094779, NIDDK 1R01DK104828, NIDDK P30-DK034987, NIDDK 1R01DK124617, NIH Ruth L. Kirschstein National Research Service Award Individual Predoctoral Fellowship (1F31DK122704), NIH T32 Genetics NIGMS Training Grant (T32-GM007092-43), NIH T32 Translational Medicine Training Grant (T32-GM122741), NIH T32 Gastroenterology Research Training Grant (T32-DK007737), and Crohn’s and Colitis Foundation Student Research Fellowship Award, and Gnotobiotic Animal Facility.

Objectives Reference values for placental weights correlated with gestational age are used in surgical pathology. Most reference values were established for fresh placentas. Some laboratories routinely fix all placentas, bringing into question the accuracy of the reference weight values. We wanted to determine the impact of fixation on placental weight. Methods One hundred placentas from uncomplicated pregnancies were weighed in the fresh state, after removal of the cord and membranes. After fixation in formalin for 1 day and 5 days, the placentas were reweighed. The change in weight for each placenta was analyzed by a two-tailed paired t test. Results Statistically, a small but significant gain in weight occurred after 24 hours (3.7%, P << .001), and there was no significant change identified in the additional 4 days (P = .51). Nine placentas lost weight with fixation; the weight of four was unchanged. Conclusions We consider formalin fixation to add a statistically significant but clinically negligible amount of weight to the placenta.

Structural chromosomal rearrangements leading to gene fusions are strong driver mutations in a variety of tumors. Identification of specific gene fusions can be essential for distinguishing benign from malignant conditions and for recognizing specific subtypes of neoplasms that can have different management and prognosis. Rapid identification of gene fusions is particularly critical for patients with acute leukemia who cannot wait more than a few days before initiating treatment and for whom treatment can be dramatically different depending on the leukemia subtype. We have developed an assay for rapid detection of oncogenic gene fusions (within 24 hours) that takes advantage of the long reads and real-time data generation of the Oxford Nanopore MinION sequencing system. By using a modification of the anchored multiplex PCR method for library construction, we confidently identified BCR-ABL1 fusion transcripts, with >100 reads within 15 minutes of sequencing. By using formalin-fixed, paraffin-embedded specimens routinely tested in our clinical molecular laboratory, fusions were successfully identified within 5 hours from acquisition of Illumina-ready libraries and 30 minutes of sequencing initiation, including cases diluted to a tumor fraction of 5%. In conclusion, we have developed a nanopore-based sequencing assay that can decrease turnaround time for detection of fusion oncogenes and may be a valid approach for laboratories with low specimen volume and for cases in need of rapid results.

Cancer patients undergoing therapeutic radiation routinely develop injury of the adjacent gastrointestinal (GI) tract mucosa due to treatment. To reduce radiation dose to critical GI structures including the rectum and oral mucosa, 3D‐printed GI radioprotective devices composed of high‐Z materials are generated from patient CT scans. In a radiation proctitis rat model, a significant reduction in crypt injury is demonstrated with the device compared to without (p, One of the most common side effects of radiation therapy is injury to the gastrointestinal (GI) tract. To reduce radiation‐induced injury, personalized 3D‐printed radioprotective devices made of high‐Z materials are developed that are shown to reduce radiation injury in two animal models, improve clinically significant dosimetry in at‐risk GI tissue in human cancer patients, and are more cost‐effective compared to standard‐of‐care therapy.

Two major molecular pathways of colorectal carcinogenesis, chromosomal instability (CIN) and microsatellite instability (MSI), are considered to be mutually exclusive. Distinguishing CIN from MSI-high tumors has considerable therapeutic implications, because patients with MSI-high tumors can derive considerable benefit from immune checkpoint inhibitors, and tumors that evolved through the CIN pathway do not respond to these agents. Familial adenomatous polyposis (FAP) is a genetic syndrome that is defined by a mutation in the APC gene and is thought to lead to carcinogenesis through the CIN pathway. Here, we report a case of a young woman with FAP who was treated for medulloblastoma as a child and developed advanced MSI-high colon cancer as a young adult. Her response to second-line immunotherapy enabled resection of her colon cancer, and she is free of disease >10 months after surgery. This case highlights the potential for overlap between the CIN and MSI carcinogenic pathways and associated therapeutic implications.

Improved tools have led to a burgeoning understanding of lung regeneration in mice, but it is not yet known how these insights may be relevant to acute lung injury in humans. We report in detail two cases of fulminant idiopathic acute lung injury requiring extracorporeal membrane oxygenation in previously healthy young adults with acute respiratory distress syndrome, one of whom required lung transplantation. Biopsy specimens showed diffuse alveolar injury with a striking paucity of alveolar epithelial regeneration, rare hyaline membranes, and diffuse contiguous airspace lining by macrophages. This novel constellation was termed diffuse alveolar injury with delayed epithelization. In addition, mirroring data from murine models of lung injury/regeneration, peribronchiolar basaloid pods (previously described as squamous metaplasia) and ciliated bronchiolarization were identified in these patients and in 39% of 57 historical cases with diffuse alveolar damage. These findings demonstrate a common and clinically relevant human disease correlate for murine models of severe acute lung injury. Evidence suggests that peribronchiolar basaloid pods and bronchiolarization are related spatially and temporally and likely represent overlapping sequential stages of the response to severe distal airway injury.

Genetic diversity among metastases is poorly understood but contains important information about disease evolution at secondary sites. Here we investigate inter- and intra-lesion heterogeneity for two types of metastases that associate with different clinical outcomes: lymph node and distant organ metastases in human colorectal cancer. We develop a rigorous mathematical framework for quantifying metastatic phylogenetic diversity. Distant metastases are typically monophyletic and genetically similar to each other. Lymph node metastases, in contrast, display high levels of inter-lesion diversity. We validate these findings by analyzing 317 multi-region biopsies from an independent cohort of 20 patients. We further demonstrate higher levels of intra-lesion heterogeneity in lymph node than in distant metastases. Our results show that fewer primary tumor lineages seed distant metastases than lymph node metastases, indicating that the two sites are subject to different levels of selection. Thus, lymph node and distant metastases develop through fundamentally different evolutionary mechanisms. Analysis of metastases using a mathematical framework and multi-region sampling data shows that lymph node metastases have higher levels of intratumor heterogeneity than distant metastases, and that these form via different evolutionary mechanisms.

B cell lymphoma and melanoma harbor recurrent mutations in the gene encoding the EZH2 histone methyltransferase (EZH2), but the carcinogenic role of these mutations is unclear. Here we describe a mouse model in which the most common somatic Ezh2 gain-of-function mutation (EZH2(Y646F) in human; Ezh2(Y641F) in mouse) is conditionally expressed. Expression of Ezh2(Y641F) in mouse B cells or melanocytes caused high-penetrance lymphoma or melanoma, respectively. Overexpression of the anti-apoptotic protein Bcl2, but not the oncoprotein Myc, or loss of the tumor suppressor protein p53 (encoded by Trp53 in mice) further accelerated lymphoma progression. Expression of the mutant Braf but not the mutant Nras oncoprotein further accelerated melanoma progression. Although expression of Ezh2(Y641F) globally increased the abundance of trimethylated Lys27 of histone H3 (H3K27me3), it also caused a widespread redistribution of this repressive mark, including a loss of H3K27me3 that was associated with increased transcription at many loci. These results suggest that Ezh2(Y641F) induces lymphoma and melanoma through a vast reorganization of chromatin structure, inducing both repression and activation of polycomb-regulated loci.

INTRODUCTION: The expression of LGR5, a known stem cell marker, is poorly understood in Barrett's esophagus (BE) and related neoplasia. The aim of this study was to evaluate LGR5 in BE and related neoplasia and to evaluate its utility as a potential biomarker of progression to advanced neoplasia. METHODS: We evaluated total 137 patients, including 119 with BE and 18 with normal gastroesophageal mucosa for expression of LGR5 using RNA in situ hybridization; this also included 28 progressors and 30 nonprogressors. The LGR5 stain was evaluated using 1 qualitative and 2 quantitative parameters, using manual and automated platforms. RESULTS: Surface LGR5 expression was mainly seen in high-grade dysplasia (12/18) compared with low-grade dysplasia (1/8) and nondysplastic BE (0/17) (P < 0.0001). In contrast to nondysplastic BE, low- and high-grade dysplasia showed a higher percentage of mean number of LGR5-positive crypts per patient (P < 0.0001) and an increase in the mean number of LGR5 transcripts per cell (P < 0.0001). The mean percentage of LGR5-positive crypts per patient and the mean number of LGR5 transcripts per cell were also significantly higher in nondysplastic BE from progressor compared with nonprogressor (P < 0.0001, P = 0.014). The sensitivity and specificity of LGR5 for distinguishing progressor from nonprogressor were 50% and 87%, respectively. DISCUSSION: BE-related advanced neoplasia shows an expansion of the LGR5-positive cellular compartment, supporting its role as a stem cell marker in this disease. Quantitative LGR5 expression and surface epithelial reactivity are novel biomarkers of increased risk of progression to advanced neoplasia in BE.

Esophageal squamous cell carcinoma (ESCC) is endemic in regions of sub-Saharan Africa (SSA), where it is the third most common cancer. Here, we describe whole-exome tumor/normal sequencing and RNA transcriptomic analysis of 59 patients with ESCC in Malawi. We observed similar genetic aberrations as reported in Asian and North American cohorts, including mutations of TP53, CDKN2A, NFE2L2, CHEK2, NOTCH1, FAT1, and FBXW7. Analyses for nonhuman sequences did not reveal evidence for infection with HPV or other occult pathogens. Mutational signature analysis revealed common signatures associated with aging, cytidine deaminase activity (APOBEC), and a third signature of unknown origin, but signatures of inhaled tobacco use, aflatoxin and mismatch repair were notably absent. Based on RNA expression analysis, ESCC could be divided into 3 distinct subtypes, which were distinguished by their expression of cell cycle and neural transcripts. This study demonstrates discrete subtypes of ESCC in SSA, and suggests that the endemic nature of this disease reflects exposure to a carcinogen other than tobacco and oncogenic viruses.

NRAS mutation at codons 12, 13, or 61 is associated with transformation; yet, in melanoma, such alterations are nearly exclusive to codon 61. Here, we compared the melanoma susceptibility of an NrasQ61R knock-in allele to similarly designed KrasG12D and NrasG12D alleles. With concomitant p16INK4a inactivation, KrasG12D or NrasQ61R expression efficiently promoted melanoma in vivo, whereas NrasG12D did not. In addition, NrasQ61R mutation potently cooperated with Lkb1/Stk11 loss to induce highly metastatic disease. Functional comparisons of NrasQ61R and NrasG12D revealed little difference in the ability of these proteins to engage PI3K or RAF. Instead, NrasQ61R showed enhanced nucleotide binding, decreased intrinsic GTPase activity, and increased stability when compared with NrasG12D. This work identifies a faithful model of human NRAS-mutant melanoma, and suggests that the increased melanomagenecity of NrasQ61R over NrasG12D is due to heightened abundance of the active, GTP-bound form rather than differences in the engagement of downstream effector pathways. Significance: This work explains the curious predominance in human melanoma of mutations of codon 61 of NRAS over other oncogenic NRAS mutations. Using conditional “knock-in” mouse models, we show that physiologic expression of NRASQ61R, but not NRASG12D, drives melanoma formation. Cancer Discov; 4(12); 1418–29. ©2014 AACR. This article is highlighted in the In This Issue feature, p. 1355

Circular RNA transcripts were first identified in the early 1990s but knowledge of these species has remained limited, as their study has been difficult through traditional methods of RNA analysis. Now, novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have allowed comprehensive studies of circular RNA species. Recent studies have revealed thousands of endogenous circular RNAs (circRNAs) in mammalian cells, some of which are highly abundant and evolutionarily conserved. Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested. Therefore, study of this class of non-coding RNAs has potential implications for therapeutic and research applications. We believe the key future challenge to the field will be to understand the regulation and function of these unusual molecules.

Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma. As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in hepatocellular carcinoma. We performed whole exome sequencing on 87 hepatocellular carcinomas and matched normal adjacent tissues to an average coverage of 59x. The overall mutation rate was roughly 2.5 mutations per Mb, with a median of 45 non-synonymous mutations that altered the amino acid sequence (range 2 to 381). We found recurrent mutations in several genes with high transcript levels: TP53 (20%), CTNNB1 (15%), IGSF3 (9%) and KEAP1 (8%). Significantly affected gene families include the nucleotide-binding domain and leucine rich repeat containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. Clinical data demonstrated significantly decreased disease-free survival in tumors with p53 mutations (p=0.006) and a trend was observed towards higher rates of recurrence and shorter disease free survival among MLL mutation carriers. The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of hepatocellular carcinoma. The elucidation of genetic changes in HCC will enhance our understanding of hepatocarcinogenesis and influence disease prognosis and therapy.

Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a “backsplice”) and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.

Genome-Wide Association studies (GWAS) offer an unbiased means to understand the genetic basis of traits by identifying single nucleotide polymorphisms (SNPs) linked to causal variants of complex phenotypes. GWAS have identified a host of susceptibility SNPs associated with many important human diseases, including diseases associated with aging. In an effort to understand the genetics of broad resistance to age-associated diseases (i.e., 'wellness'), we performed a meta-analysis of human GWAS. Toward that end, we compiled 372 GWAS that identified 1775 susceptibility SNPs to 105 unique diseases and used these SNPs to create a genomic landscape of disease susceptibility. This map was constructed by partitioning the genome into 200 kb 'bins' and mapping the 1775 susceptibility SNPs to bins based on their genomic location. Investigation of these data revealed significant heterogeneity of disease association within the genome, with 92% of bins devoid of disease-associated SNPs. In contrast, 10 bins (0.06%) were significantly (P < 0.05) enriched for susceptibility to multiple diseases, 5 of which formed two highly significant peaks of disease association (P < .0001). These peaks mapped to the Major Histocompatibility (MHC) locus on 6p21 and the INK4/ARF (CDKN2a/b) tumor suppressor locus on 9p21.3. Provocatively, all 10 significantly enriched bins contained genes linked to either inflammation or cellular senescence pathways, and SNPs near regulators of senescence were particularly associated with disease of aging (e.g., cancer, atherosclerosis, type 2 diabetes, glaucoma). This analysis suggests that germline genetic heterogeneity in the regulation of immunity and cellular senescence influences the human healthspan.

Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in gliomas, myeloid leukemias, chondrosarcomas, and thyroid cancer. We discovered IDH1 and IDH2 mutations in 34 of 326 (10%) intrahepatic cholangiocarcinomas. Tumor with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine (5hmC) and higher 5-methylcytosine (5mC) levels, as well as increased dimethylation of histone H3K79. Mutations in IDH1 or IDH2 were associated with longer overall survival (p = 0.028) and were independently associated with a longer time to tumor recurrence after intrahepatic cholangiocarcinoma resection in multivariate analysis (p = 0.021). IDH1 and IDH2 mutations are significantly associated with increased levels of p53 in intrahepatic cholangiocarcinomas, but no mutations in the p53 gene were found, suggesting that mutations in IDH1 and IDH2 may cause a stress that leads to p53 activation. We identified 2,309 genes that were significantly hypermethylated in 19 cholangiocarcinomas with mutations in IDH1 or IDH2, compared with cholangiocarcinomas without these mutations. Hypermethylated CpG sites were significantly enriched in CpG shores and upstream of transcription start sites, suggesting a global regulation of transcriptional potential. Half of the hypermethylated genes overlapped with DNA hypermethylation in IDH1-mutant gliobastomas, suggesting the existence of a common set of genes whose expression may be affected by mutations in IDH1 or IDH2 in different types of tumors.

Studying development in diverse taxa can address a central issue in evolutionary biology: how morphological diversity arises through the evolution of developmental mechanisms. Two of the best-studied developmental model organisms, the arthropod Drosophila and the nematode Caenorhabditis elegans, have been found to belong to a single protostome superclade, the Ecdysozoa. This finding suggests that a closely related ecdysozoan phylum could serve as a valuable model for studying how developmental mechanisms evolve in ways that can produce diverse body plans. Tardigrades, also called water bears, make up a phylum of microscopic ecdysozoan animals. Tardigrades share many characteristics with C. elegans and Drosophila that could make them useful laboratory models, but long-term culturing of tardigrades historically has been a challenge, and there have been few studies of tardigrade development. Here, we show that the tardigrade Hypsibius dujardini can be cultured continuously for decades and can be cryopreserved. We report that H. dujardini has a compact genome, a little smaller than that of C. elegans or Drosophila, and that sequence evolution has occurred at a typical rate. H. dujardini has a short generation time, 13–14 days at room temperature. We have found that the embryos of H. dujardini have a stereotyped cleavage pattern with asymmetric cell divisions, nuclear migrations, and cell migrations occurring in reproducible patterns. We present a cell lineage of the early embryo and an embryonic staging series. We expect that these data can serve as a platform for using H. dujardini as a model for studying the evolution of developmental mechanisms.

Summary We describe the landscape of genomic alterations in cutaneous melanomas through DNA, RNA, and protein-based analysis of 333 primary and/or metastatic melanomas from 331 patients. We establish a framework for genomic classification into one of four subtypes based on the pattern of the most prevalent significantly mutated genes: mutant BRAF , mutant RAS , mutant NF1 , and Triple-WT (wild-type). Integrative analysis reveals enrichment of KIT mutations and focal amplifications and complex structural rearrangements as a feature of the Triple-WT subtype. We found no significant outcome correlation with genomic classification, but samples assigned a transcriptomic subclass enriched for immune gene expression associated with lymphocyte infiltrate on pathology review and high LCK protein expression, a T cell marker, were associated with improved patient survival. This clinicopathological and multi-dimensional analysis suggests that the prognosis of melanoma patients with regional metastases is influenced by tumor stroma immunobiology, offering insights to further personalize therapeutic decision-making.

Somatic sequencing of cancers has produced new insight into tumorigenesis, tumor heterogeneity, and disease progression, but the vast majority of genetic events identified are of indeterminate clinical significance. Here, we describe a NextGen sequencing approach to fully analyzing 248 genes, including all those of known clinical significance in melanoma. This strategy features solution capture of DNA followed by multiplexed, high-throughput sequencing and was evaluated in 31 melanoma cell lines and 18 tumor tissues from patients with metastatic melanoma. Mutations in melanoma cell lines correlated with their sensitivity to corresponding small molecule inhibitors, confirming, for example, lapatinib sensitivity in ERBB4 mutant lines and identifying a novel activating mutation of BRAF. The latter event would not have been identified by clinical sequencing and was associated with responsiveness to a BRAF kinase inhibitor. This approach identified focal copy number changes of PTEN not found by standard methods, such as comparative genomic hybridization (CGH). Actionable mutations were found in 89% of the tumor tissues analyzed, 56% of which would not be identified by standard-of-care approaches. This work shows that targeted sequencing is an attractive approach for clinical use in melanoma.

Inexpensive de novo genome sequencing, particularly in organisms with small genomes, is now possible using several new sequencing technologies. Some of these technologies such as that from Illumina's Solexa Sequencing, produce high genomic coverage by generating a very large number of small reads (∼30 bp). While prior work shows that partial assembly can be performed by k-mer extension in error-free reads, this algorithm is unsuccessful with the sequencing error rates found in practice. We present VCAKE (Verified Consensus Assembly by K-mer Extension), a modification of simple k-mer extension that overcomes error by using high depth coverage. Though it is a simple modification of a previous approach, we show significant improvements in assembly results on simulated and experimental datasets that include error.Availability: http://152.2.15.114/~labweb/VCAKEContact: william.jeck@gmail.com

Objectives: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients prospectively enrolled on an IRB-approved institutional tumor sequencing initiative (UNCseq) and correlate these with clinical factors. Methods: Snap-frozen and FFPE tissue samples were collected from patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1). Results: Data was collected from 109 EC patients. Thirty-six tumors (33.0%), 34 of which were endometrioid, had >2 mutations between PIK3CA and PIK3R1. 20 patients had multiple PIK3CA mutations (18.3%) and 11 had multiple PIK3R1 mutations (10.1%). In all but 5 cases, multiple mutations of PIK3R1 or PIK3CA were coincident with PTEN mutation (p = 0.018). There were strong correlations between PI3K pathway mutations and tumor grade and stage. High stage tumors (stage >2) were less likely (5/36) than stage 1 tumors (31/73) to have multiple PI3K mutations (p = 0.0056). Though not reaching significance, grade 3 tumors (8/37) were less likely than low-grade tumors (28/72) to demonstrate multiple PI3K mutations (p = 0.11). Of the 10 serous ECs, none exhibited multiple PI3K mutations. Conclusions: There is a strong relationship between mutated PIK3 pathway genes and clinically relevant tumor biology in ECs. Multiple mutations in the PIK3 pathway are common, suggesting that PI3K pathway inhibition in EC may need to be directed against PIK3CA and PIK3R1 simultaneously. These multiply mutated tumors are strongly linked to lower stage and grade tumors with concurrent PTEN mutations. We posit that initially PTEN mutant clones may give rise to subclonal populations with various PI3K mutations which may be relatively indolent until additional driver mutations are acquired such that one subpopulation predominates, resulting in higher grade and stage ECs. Citation Format: Dario R. Roque, Will R. Jeck, David N. Hayes, Arthur M. Dizon, Leslie Clark, Stuart Pierce, Weiya Z. Wysham, Tara Castellano, Paola A. Gehrig, Victoria Bae-Jump. Multiple PI3K pathway mutations predominate in low stage endometrial carcinomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1852.

Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma (HCC). As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in HCC. We performed whole-exome sequencing on 87 HCCs and matched normal adjacent tissues to an average coverage of 59×. The overall mutation rate was roughly two mutations per Mb, with a median of 45 nonsynonymous mutations that altered the amino acid sequence (range, 2-381). We found recurrent mutations in several genes with high transcript levels: TP53 (18%); CTNNB1 (10%); KEAP1 (8%); C16orf62 (8%); MLL4 (7%); and RAC2 (5%). Significantly affected gene families include the nucleotide-binding domain and leucine-rich repeat-containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors.The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of HCC.

Genome-Wide Association studies (GWAS) offer an unbiased means to understand the genetic basis of traits by identifying single nucleotide polymorphisms (SNPs) linked to causal variants of complex phenotypes. GWAS have identified a host of susceptibility SNPs associated with many important human diseases, including diseases associated with aging. In an effort to understand the genetics of broad resistance to age-associated diseases (i.e. ‘wellness’), we performed a meta-analysis of human GWAS. Toward that end, we compiled 372 GWAS that identified 1,775 susceptibility SNPs to 105 unique diseases and used these SNPs to create a genomic landscape of disease susceptibility. This map was constructed by partitioning the genome into 200 kb ‘bins’ and mapping the 1,775 susceptibility SNPs to bins based on their genomic location. Investigation of these data revealed significant heterogeneity of disease association within the genome, with 92% of bins devoid of disease-associated SNPs. In contrast, 10 bins (0.06%) were significantly (p

We developed a novel approach for de novo genome assembly using only sequence data from high-throughput short read sequencing technologies. By combining data generated from 454 Life Sciences (Roche) and Illumina (formerly known as Solexa sequencing) sequencing platforms, we reliably assembled genomes into large scaffolds at a fraction of the traditional cost and without use of a reference sequence. We applied this method to two isolates of the phytopathogenic bacteria Pseudomonas syringae. Sequencing and reassembly of the well-studied tomato and Arabidopsis pathogen, PtoDC3000, facilitated development and testing of our method. Sequencing of a distantly related rice pathogen, Por1_6, demonstrated our method's efficacy for de novo assembly of novel genomes. Our assembly of Por1_6 yielded an N50 scaffold size of 531,821 bp with >75% of the predicted genome covered by scaffolds over 100,000 bp. One of the critical phenotypic differences between strains of P. syringae is the range of plant hosts they infect. This is largely determined by their complement of type III effector proteins. The genome of Por1_6 is the first sequenced for a P. syringae isolate that is a pathogen of monocots, and, as might be predicted, its complement of type III effectors differs substantially from the previously sequenced isolates of this species. The genome of Por1_6 helps to define an expansion of the P. syringae pan-genome, a corresponding contraction of the core genome, and a further diversification of the type III effector complement for this important plant pathogen species.

Affiliations des auteurs : cf page 216 de l'article; International audience; Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

e16522 Background: Endometrial cancers (ECs) frequently harbor mutations in PI3K pathway genes. Our objective was to determine the mutation frequency of the PIK3 pathway in the tumors of EC patients enrolled on UNCseq and correlate these findings with clinical outcomes. Methods: Snap-frozen and FFPE tissue samples were collected from EC patients enrolled on UNCseq (NCT01457196). DNA was isolated using the Puregene DNA purification system, and Illumina libraries were prepared separately from tumor and a matched normal sample from each patient. Relevant targets, were enriched by a custom designed Agilent SureSelect hybrid capture enrichment library using standard protocols. Samples were sequenced on Illumina HiSeq machines in a variety of formats. Statistical analysis of clinical correlates was performed in R (v 3.1.1). Results: Data was collected from 73 EC patients. Fifteen tumors (20.5%), 14 of which were endometrioid, had 2 or more mutations in PIK3CA or PIK3R1. Of 73 patients, 6 patients had 2 PIK3CA m...

Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma. As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in hepatocellular carcinoma. We performed whole exome sequencing on 87 hepatocellular carcinomas and matched normal adjacent tissues to an average coverage of 59x. The overall mutation rate was roughly 2.5 mutations per Mb, with a median of 45 non-synonymous mutations that altered the amino acid sequence (range 2 to 381). We found recurrent mutations in several genes with high transcript levels: TP53 (20%), CTNNB1 (15%), IGSF3 (9%) and KEAP1 (8%). Significantly affected gene families include the nucleotide-binding domain and leucine rich repeat containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. Clinical data demonstrated significantly decreased disease-free survival in tumors with p53 mutations (p=0.006) and a trend was observed towards higher rates of recurrence and shorter disease free survival among MLL mutation carriers. The NFE2L2-KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of hepatocellular carcinoma. The elucidation of genetic changes in HCC will enhance our understanding of hepatocarcinogenesis and influence disease prognosis and therapy. Citation Format: Sean P. Cleary, William R. Jeck, Xiaobei Zhao, Sara R. Selitsky, Gleb L. Savich, Ting-Xu Tan, Michael C. Wu, Gad Getz, Michael S. Lawrence, Joel S. Parker, Jinyu Li, Scott Powers, Hyeja Kim, Sandra E. Fischer, Maha Guindi, Anand Ghanekar, Derek Y. Chiang. Identification of driver genes in hepatocellular carcinoma by exome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4598. doi:10.1158/1538-7445.AM2013-4598

In human cancer, N-RAS mutations resulting in constitutive, oncogenic signaling are predominately localized to codons 12, 13 or 61. Traditionally, activating RAS mutations have been considered oncogenically equivalent, yet recent studies suggest important clinical distinctions between colon cancers containing K-RAS codon 12 vs. codon 13 mutations. More than 20% of human melanomas harbor N-RAS mutations, the vast majority of which target codon 61, as opposed to N-RAS mutations of codon 12 or 13, which are common in other tumor types. To address this issue, we characterized syngeneic knock-in mouse models conditionally expressing either N-RasG12D or N-RasQ61R under the control of the endogenous locus. In primary melanocyte cultures, activation of either N-Ras allele reduced cellular proliferation even in the presence of concomitant p16INK4a deletion. Correspondingly, melanocyte-specific N-RasG12D expression combined with p16INK4a loss failed to efficiently promote melanoma formation in mice. In contrast, melanocyte-specific N-RasQ61R induction readily combined with p16INK4a loss to promote melanoma formation in vivo with short latency and high penetrance. These results provide a first murine model of melanoma featuring knock-in N-Ras mutationQ61R, and suggest that the preference for codon 61 mutations in human tumors is conserved in mice. Citation Format: Christin Burd, William R. Jeck, Kailing Fu, Kelly S. Clark, Jessie C. Xiong, George P. Souroullas, Norman E. Sharpless. Differential oncogenicity of N-RAS mutations in melanoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2013-1087

Cholangiocarcinomas are rare but aggressive cancers, with an increasing incidence and a dismal 5-year survival rate of less than 5%. Mutations in the genes encoding isocitrate dehydrogenase, IDH1 and IDH2, have been reported in ∼70% of gliomas, ∼20% of myeloid leukemias, ∼56% of chondrosarcomas, and ∼10% of melanomas. We discovered IDH1 and IDH2 mutations in 29 of 305 (9%) intrahepatic cholangiocarcinomas. Tumors with mutations in IDH1 or IDH2 had lower 5-hydroxymethylcytosine and higher 5-methylcytosine levels, as well as increased dimethylation of histone H3K79. Mutations in IDH1 or IDH2 were independently associated with longer time to recurrence of cholangiocarcinoma in multivariate analysis (p = 0.046). We identified 2,267 genes that were significantly hypermethylated in tumors with mutations in IDH1 or IDH2. Hypermethylated CpG sites were significantly enriched in CpG shores and in the 1500 bp upstream of annotated transcription start sites, suggesting a global regulation of transcriptional potential. Gene expression profiling of 7 IDH1/2-mutant and 20 IDH1/2-wildtype tumors revealed increased expression of citric acid cycle and oxidative phosphorylation enzymes, as well as decreased expression of cytoskeleton organization genes. Integrated analyses of DNA methylation and gene expression identified interleukin-6 and several of its downstream targets as candidates for silencing by DNA methylation. Conclusions: Cholangiocarcinomas with mutations in IDH1 or IDH2 represent a molecular subclass with distinct signaling pathway alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1058. doi:1538-7445.AM2012-1058