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BackgroundPediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions.ResultsOur ensemble fusion detection approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and AWS serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a “known fusion list” prevents failure to report known pathogenic events. We have employed the ensemble fusion-calling pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient’s medical record, both known and novel fusions provided medically meaningful information.ConclusionsOur ensemble fusion detection pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

Retinoblastoma is a childhood cancer of the retina involving germline or somatic alterations of the RB Transcriptional Corepressor 1 gene, RB1. Rare cases of sellar-suprasellar region retinoblastoma without evidence of ocular or pineal tumors have been described. A nine-month-old male presented with a sellar-suprasellar region mass. Histopathology showed an embryonal tumor with focal Flexner-Wintersteiner-like rosettes and loss of retinoblastoma protein (RB1) expression by immunohistochemistry. DNA array-based methylation profiling confidently classified the tumor as pineoblastoma group A/intracranial retinoblastoma. The patient was subsequently enrolled on an institutional translational cancer research protocol and underwent comprehensive molecular profiling, including paired tumor/normal exome and genome sequencing and RNA-sequencing of the tumor. Additionally, Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing was performed from comparator normal and disease-involved tissue to resolve complex structural variations. RNA-sequencing revealed multiple fusions clustered within 13q14.1-q21.3, including a novel in-frame fusion of RB1-SIAH3 predicted to prematurely truncate the RB1 protein. SMRT sequencing revealed a complex structural rearrangement spanning 13q14.11-q31.3, including two somatic structural variants within intron 17 of RB1. These events corresponded to the RB1-SIAH3 fusion and a novel RB1 rearrangement expected to correlate with the complete absence of RB1 protein expression. Comprehensive molecular analysis, including DNA array-based methylation profiling and sequencing-based methodologies, were critical for classification and understanding the complex mechanism of RB1 inactivation in this diagnostically challenging tumor.

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The existence of chronic typhoid carriers has been in the public eye for over 100 years in part because of the publicity around Typhoid Mary. Additionally, it has been known for decades that the gallbladder is the main site of persistence and recently that gallstones play a key role. Despite this, very little is known about the physiological conditions that allow Salmonella enterica serovar Typhi to persist in the gallbladder. In this study, we analyze the transcriptional profile of the gallbladder in a mouse model of chronic carriage. We found a shift from an early proinflammatory immune response toward a later anti-inflammatory response, which could explain the stalemate that allows Salmonella persistence. Interestingly, we found a 10-fold increase in the number of Salmonella-specific T cells in mice with gallstones. This work moves us closer to understanding the mechanistic basis of chronic carriage, with a goal toward eradication of the disease., Typhoid fever, caused primarily by Salmonella enterica serovar Typhi (S. Typhi), is a life-threatening systemic disease responsible for significant morbidity and mortality worldwide. Three to 5% of individuals infected with S. Typhi become chronic carriers due to bacterial persistence in the gallbladder. We have demonstrated that Salmonella forms biofilms on gallstones to establish gallbladder carriage. However, an in-depth molecular understanding of chronic carriage in the gallbladder, from the perspective of both the pathogen and host, is poorly defined. To examine the dynamics of the gallbladder in response to Salmonella infection, we performed transcriptional profiling in the mouse gallbladder at early (7 days) and chronic (21 days) time points. Transcriptome sequencing (RNA-Seq) revealed a shift from a Th1 proinflammatory response at 7 days postinfection (dpi) toward an anti-inflammatory Th2 response by 21 dpi, characterized by increased levels of immunoglobulins and the Th2 master transcriptional regulator, GATA3. Additionally, bioinformatic analysis predicted the upstream regulation of characteristic Th2 markers, including interleukin-4 (IL-4) and Stat6. Immunohistochemistry and fluorescence-activated cell sorter (FACS) analysis confirmed a significant increase in lymphocytes, including T and B cells, at 21 dpi in mice with gallstones. Interestingly, the levels of Salmonella-specific CD4 T cells were 10-fold higher in the gallbladder of mice with gallstones at 21 dpi. We speculate that the biofilm state allows Salmonella to resist the initial onslaught of the Th1 inflammatory response, while yet undefined events influence a switch in the host immunity toward a more permissive type 2 response, enabling the establishment of chronic infection.

Typhoid fever is caused primarily by Salmonella enterica serovar Typhi. Approximately 3% to 5% of individuals infected with S. Typhi become chronic carriers with the gallbladder (GB) as the site of persistence, as gallstones within the GB are a platform on which the bacteria form a biofilm. S. Typhi is a human-restricted pathogen; therefore, asymptomatic carriers represent a critical reservoir for further spread of disease. To examine the dynamics of the Salmonella biofilm during chronic carriage, the human gallstone (GS) environment was simulated by growing biofilms on cholesterol-coated surfaces in the presence of bile, and the transcriptional profile was determined. Some of the most highly activated genes corresponded to the curli fimbria operon, with the major structural component csgA upregulated >80-fold. The curli protein polymer is a major component of the extracellular matrix (ECM) in Salmonella biofilms. The upregulation of curli fimbriae by human bile was validated through reverse transcription-quantitative PCR (qRT-PCR), microscopy, and Western blotting. Interestingly, this activation appears human specific, as qRT-PCR showed repression of csgA in biofilms grown in mouse or ox bile. Comparative transcriptional studies of the two divergent csg operons suggest an early activation of both operons in minimal medium complemented with glucose that quickly diminishes as the biofilm matures. However, in the presence of human bile, there is a modest activation of both operons that steadily increases as the biofilm matures. Understanding the effect of the GB environment on key biofilm-associated factors can help target antibiofilm therapeutics or other preventative strategies to eradicate chronic carriage. IMPORTANCE Typhoid fever is caused by Salmonella enterica serovar Typhi, and 3% to 5% of patients become chronic gallbladder (GB) carriers (also known as “Typhoid Marys”). We have previously demonstrated a role for Salmonella biofilm formation on gallstones as a primary mechanism of carriage. In this study, we found that the important biofilm extracellular matrix component curli fimbria is induced in an in vitro human GB model system. This induction is specific to human bile and increases as the biofilm matures. We also found that the biofilm and curli regulator CsgD play a key role in this observed induction. This work further enhances our understanding biofilm-mediated chronic carriage and provides a potential target for eliminating persistent GB infection by S. Typhi.

A series of new carbon molecular sieve fibers (CMSF) was made by a two-step pyrolysis of melt extruded polyvinylidene chloride copolymer (PVDC) fibers. The PVDC fibers were first pretreated in air at low temperature to dehydrochlorinate and crosslink, and then pyrolyzed in N2 at high temperatures from 500 to 1400 °C. A high throughput kinetic adsorption tool with 48 parallel testing cells using 9 probe molecules (kinetic diameter from 3.3 A −5.5 A) was used to 1) characterize the effective micropore size and its changes, and 2) fast screen the adsorbents for several gas separations. The effective micropore size continuously shrank from ~4.9 to 3.4 A, as the final pyrolysis temperature increased from 500 to 1400 °C. The CMSF adsorbents can be tuned for several gas separations: N2/CH4, C2H4/C2H6, and C3H6/C3H8. The newly discovered CMS has 54% larger capacity and 86% larger working capacity (in 13–250 kPa pressure range) of propylene than the commercial MSC-4K CMS adsorbent. By reducing the diameter of CMSF from 100 to 50 μm, the adsorption kinetics was improved while maintaining good separation factor in the range of 12–29. Furthermore, this new CMSF adsorbent has potential to form future advanced structured adsorbents for rapid cycle pressure swing adsorption to achieve a smaller adsorbent bed.

The renal collecting duct consists of intercalated cells (ICs) and principal cells (PCs). We have previously demonstrated that collecting ducts have a role in the innate immune defense of the kidney. Transcriptomics is an important tool used to enhance systems-level understanding of cell biology. However, transcriptomics performed on whole kidneys provides limited insight of collecting duct cell gene expression, because these cells comprise a small fraction of total kidney cells. Recently we generated reporter mouse models to enrich collecting duct specific PC and ICs and reported targeted gene expression of anti-microbial peptide genes. Here we report transcriptomics on enriched ICs and PCs and performed a pilot study sequencing four single ICs. We identified 3,645 genes with increased relative expression in ICs compared to non-ICs. In comparison to non-PCs, 2,088 genes had higher relative expression in PCs. IC associated genes included the innate interleukin 1 receptor, type 1 and the antimicrobial peptide (AMP) adrenomedullin. The top predicted canonical pathway for enriched ICs was lipopolysaccharide/Interleukin 1 mediated inhibition of Retinoid X Receptor alpha function and decreased Retinoid X Receptor expression was confirmed to occur 1-hour post experimental murine UTI in ICs but not in non-ICs.

Background We report a female patient with endocrine abnormalities, hypogonadotropic hypogonadism and amazia (breasts aplasia/hypoplasia but normal nipples and areolas) in a rare syndrome: Van Maldergem syndrome (VMS). Case presentation Our patient was first evaluated at age 4 for intellectual disability, craniofacial features, and auditory malformations. At age 15, she presented with no breast development and other findings consistent with hypogonadotropic hypogonadism. At age 37, she underwent whole exome sequencing (WES) to identify pathogenic variants. WES revealed compound heterozygous variants in DCHS1 (rs145099391:G > A, p.P197L & rs753548138:G > A, p.T2334 M) [RefSeq NM_003737.3], diagnostic of Van Maldergem syndrome (VMS-1). VMS is a rare autosomal disorder reported in only 13 patients, characterized by intellectual disability, typical craniofacial features, auditory malformations, hearing loss, skeletal and limb malformations, brain abnormalities with periventricular neuronal heterotopia and other variable anomalies. Our patient had similar phenotypic abnormalities. She also had hypogonadotropic hypogonadism and amazia. Based on the clinical findings reported, two previously published patients with VMS may also have been affected by hypogonadotropic hypogonadism, but endocrine abnormalities were not evaluated or mentioned. Conclusion This case highlights an individual with VMS, characterized by compound heterozygous variants in DCHS1. Our observations may provide additional information on the phenotypic spectrum of VMS, including hypogonadotropic hypogonadism and amazia. However, the molecular genetic basis for endocrine anomalies observed in some VMS patients, including ours, remains unexplained. Electronic supplementary material The online version of this article (10.1186/s13633-017-0052-z) contains supplementary material, which is available to authorized users.

Pediatric cancers, which make up ~1% of cancer diagnoses each year, comprise distinct genomic landscapes compared to adult cancers. Pediatric cancers typically exhibit a “quiet” genome with a reduced number of somatic mutations compared to adult tumors, which tend to carry a high mutational burden. In addition, pediatric tumors are often associated with fusion events, including fusions that involve potentially targetable kinases, as well as transcription factors which can often be targeted downstream. Fusion events can occur through chromosomal rearrangements, large deletions, or insertions. These events can lead to dysregulated gene expression, and can often become a driving event in pediatric cancer. Sensitive and specific detection of these fusion events through the utilization of RNA-seq data has proven to be a difficult task, given the complexities in tumor cellularity and clonality, and the numerous false positive identifications that are typically output from single fusion callers. Additionally, there are confounding issues surrounding memory usage and compute time required for certain fusion calling algorithms. The Institute for Genomic Medicine at Nationwide Children’s Hospital has implemented an ensemble approach, utilizing 7 fusion callers: FusionMap, JAFFA, STAR-Fusion, SOAPfuse, FusionCatcher, MapSplice, and TopHat-Fusion. This approach allows us to identify fusions that have been called by at least 2 of the above algorithms, and also allows for a prioritization approach based on the number of callers that have identified a specific fusion. We have employed this approach on 67 pediatric cancer cases that we have analyzed in a collaborative Institutional Review Board approved protocol which encompasses scientists, genetic counselors, oncologists, and pathologists at Nationwide Children’s Hospital. These cases are comprised of 52 central nervous system (CNS) tumors, 13 solid tumors, and 1 hematologic tumor, to date. To analyze these cases for fusion events, total RNA-seq with ribodepletion was performed on RNA, extracted from either flash frozen or Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples, to generate libraries that were sequenced using 150bp paired-end reads. Through the employment of our ensemble fusion calling approach and a manual knowledge based filtering strategy, we have currently identified 18 clinically meaningful fusions, all of which we have confirmed in a CAP/CLIA laboratory. We have identified both known and novel fusion events, of which several have provided diagnostic value and/or provided targeted treatment options for patients. In conclusion, this method has the potential to offer a streamlined approach to uncover driver fusions in cancer, while also providing additional diagnostics and an opportunity to identify targeted treatment options for patients in a clinical setting. Citation Format: Stephanie LaHaye, Kyle Voytovich, James Fitch, Sean McGrath, Anthony Miller, Amy Wetzel, Vincent Magrini, Elaine R. Mardis, Richard K. Wilson, Peter White, Catherine E. Cottrell. Utilization of an ensemble approach for identification of driver fusions in pediatric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1651.

Cancer treatment strategies targeting the tumor microenvironment and corresponding immune cell infiltrate (i.e., immunotherapy) have recently proven effective against many solid tumors. In pediatric brain cancers, there is little known about the immune tumor microenvironment. We aim to characterize immune cell populations of pediatric brain tumors, which will better inform us about immune cell infiltration in different brain cancer subtypes and may indicate potential for patient response to immunotherapy. The Institute for Genomic Medicine at Nationwide Children’s Hospital has recently initiated a translational protocol to evaluate tumor specimens from pediatric patients with rare or refractory brain cancers in a focused N-of-1 manner. We performed RNA-seq and NanoString™ PanCancer Immune expression profiling on extracted tumor RNA from 22 patients comprising eight distinct brain cancer subtypes. We used CIBERSORT algorithm for deconvolution of RNA-seq global gene expression data and NanoString™ expression profiling to investigate methods of quantifying tumor-infiltrating leukocytes (TIL) and expression of immune genes. Our analyses identified a subset of pediatric brain tumors with evidence of immune infiltration. Using CIBERSORT, four gliomas were predicted to harbor significant TIL populations. Overall, we observed high correlation (Pearson R2 = 0.884) between expression values derived from RNA-seq and NanoString™ methods. Similarly, the two methods produced comparable predictions for TIL composition in the glioma tumors. Further genomic analysis identified one patient, with a diagnosis of glioblastoma and Neurofibromatosis type 1 (NF1), with marked infiltration of macrophages and neutrophils. Overexpression of tumor-associated macrophage/microglia genes (CD14, CD163, CD68, ITGAM, PTPRC) and other immune markers (PD-L1, CCL2/MCP-1, IL1B) was observed relative to all other samples. More specifically, overexpression of CD204, CD206, and CD163 suggested the infiltrating macrophages represent the M2/protumor phenotype. CIBERSORT allows more “granularity” in macrophage population prediction and indicated an equal representation of M2 and M0/uncommitted macrophages. Given the M2 phenotype, it is likely that the infiltrating macrophages contributed to tumor progression and radiation resistance observed in this patient. If confirmed, the presence of M0 macrophages and/or overexpression of PD-L1 could indicate potential for immunotherapy in this patient (e.g., oncolytic viral therapy, strategies focusing on M0 to M1/antitumor polarization, or combination targeted therapy with PD-L1 inhibitor). In summary, we identified a patient with progressive glioblastoma tumor growth and significant macrophage tumor infiltration as a likely candidate for immunotherapy. Citation Format: Katherine E. Miller, Kathleen Schieffer, James Fitch, Vincent Magrini, Amy Wetzel, Anthony R. Miller, Daniel R. Boue, Jeffrey Leonard, Jonathan L. Finlay, Diana S. Osorio, Mohamed S. AbdelBaki, Christopher R. Pierson, Annie Drapeau, Jonathan Pindrik, Kristen Leraas, Elizabeth Varga, Devon Dishman, Lauren Shoemaker, Nicole Ross, Jeremy Pitts, Julie Gastier-Foster, Peter White, Catherine E. Cottrell, Richard K. Wilson, Elaine R. Mardis. Expression profiling-based characterization of immune cell populations in pediatric brain cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4551.

Medulloblastoma is a pediatric embryonal tumor that can be classified into four molecular subgroups, each derived from a different progenitor cell. It is estimated that about 30-40% of patients will relapse, typically with recurrence at the primary site and of the same molecular subgroup. We present paired tumor/normal genomic analysis of an 18 year-old male who presented with non-Wnt/non-SHH medulloblastoma at age 12 and relapsed with metastatic disease of the falx cerebri 3 years later. Combination surgery, chemotherapy, and radiation were used in treatment of the primary and recurrent tumor. At a timepoint 6 years from original diagnosis, the patient presented with a cerebellar tumor histologically described as “consistent with recurrent medulloblastoma” with comment recommending genomics to confirm. The diagnosis was made based on near identifical morphology and retention of Neu-N and Synaptophysin in the tumor (confirmed by subsequent genetic analysis). The primary tumor and the tumor occurring 6 years after the primary diagnosis were analyzed by whole exome sequencing (blood and tumor tissues) to assess for germline variants, somatic mutation, and copy number variation. We observed no pathogenic germline variants in cancer predisposition genes. The tumor mutational profiles were distinct, with only 6 (1.8%) shared somatic variants between tumors. Specimen provenance was verified by germline variation and SRY coverage. Two targetable mutations within the RAS-MAPK pathway (PTPN11 p.Glu76Lys and PIK3CA p.Gly1007Arg) were present only in the new CNS tumor. Although the primary tumor harbored isochromosome 17q and a gain of chromosome 4, these somatic chromosomal aberrations were not detected in the new CNS tumor. RNA-seq was performed on both tumors and compared to pediatric CNS tumors from the University of California Santa Cruz Treehouse Initiative (n=434). The primary tumor clustered with the medulloblastoma patients by principal component analysis while the new CNS tumor clustered with a group of gliomas and non-medulloblastoma embryonal tumors. The primary tumor displayed evidence of overexpression of Group 4 medulloblastoma genes (e.g. EOMES, RBM24, SNCAIP, and UNC5D). These genes were not overexpressed in the new CNS tumor. Enrichment of genes commonly found in gliomas (e.g. BCAN, CHI3L2, PDGFRA, and SOX2) were noted in the new CNS tumor only. In summary, tumor genomic profiling of a primary medulloblastoma and the new CNS tumor arising 6 years later revealed two distinct sets of somatic mutations suggestive of second malignancy rather than recurrence in this patient. While second malignancy in the setting of medulloblastoma is a rare event, it has been documented, both in a time period consistent with that described in our patient and in the form of glioma. Thus, tumor profiling refined diagnosis in this patient allowing for a more accurate assessment of treatment and management options. Citation Format: Kathleen M. Schieffer, Katherine E. Miller, Daniel R. Boue, Daniel C. Koboldt, Patrick Brennan, Benjamin J. Kelly, Gregory Wheeler, Vincent Magrini, Amy Wetzel, Elizabeth Varga, Devon Dishman, Kristen Leraas, Vibhuti Agarwal, Mohamed S. AbdelBaki, Jonathan L. Finlay, Jeffrey R. Leonard, Peter White, Julie M. Gastier-Foster, Catherine E. Cottrell, Elaine R. Mardis, Richard K. Wilson. Molecular profiling identifies a second malignancy in a patient with medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 484.

While advances in genome sequencing technology make population-scale genomics a possibility, current approaches for analysis of these data rely upon parallelization strategies that have limited scalability, complex implementation and lack reproducibility. Churchill, a balanced regional parallelization strategy, overcomes these challenges, fully automating the multiple steps required to go from raw sequencing reads to variant discovery. Through implementation of novel deterministic parallelization techniques, Churchill allows computationally efficient analysis of a high-depth whole genome sample in less than two hours. The method is highly scalable, enabling full analysis of the 1000 Genomes raw sequence dataset in a week using cloud resources. http://churchill.nchri.org/. Electronic supplementary material The online version of this article (doi:10.1186/s13059-014-0577-x) contains supplementary material, which is available to authorized users.

The Dow Chemical Company's Aluminum-Boron Carbide Composite (AlBC) is a material which has the density of aluminum combined with significantly improved stiffness, hardness and ability to perform in continuous use at temperatures substantially higher than aluminum. AlBC composite is electrically conductive, has high compressive strength and high heat capacity. AlBC represents a broad family of materials which can be tailored during the manufacturing process to the material requirements of different applications. AlBC composites span the composition range from ceramics to metals in the ratios from 99:1 to 1:99. In the upper range of ceramic content, the properties of AlBC composites are similar to those of ceramics with improved toughness and in the lower range similar to metals but with improved hardness, wear resistance and higher temperature resistance. This paper discusses the design and properties of AlBC materials in the intermediate compositional range between ∼ 65% and 90% ceramic content. The composition at the lower boundary of this range is an alternative to conventional aluminum metal-matrix composite (Al-MMC) for applications that require performance beyond the useful range of typical Al-MMC. The upper boundary composition is an alternative to cast iron in weight-sensitive applications that require temperature stability up to 500-600°C.