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Highly pure biogenic silica can be obtained from rice husk ash if agglomeration and ash melting during combustion can be avoided. In the present study, the effects of fuel pretreatment and combusti...

In the short- and mid-term perspective, drastic measures for the reduction of anthropogenic emissions including extensive decarbonization of the residential and industrial heating sector have to be implemented. To replace fossil fuels, solid biogenic residues and wastes will have to be increasingly utilized. Compared to clean woody biomass, these biomass assortments are commonly characterized by higher Si and alkaline metal contents recognized as major driver for low ash melting temperatures resulting in elevated risk of bottom ash slagging. To facilitate the prediction of bottom ash slagging during combustion, several fuel indices have been proposed. Based on empirical correlations with parameters relevant for slagging behavior, e.g., ash melting temperatures or slag fraction of the bottom ash, these fuel indices were subsequently enhanced and adapted for an increasing range of biomass fuel characteristics. In this study, analysis data of 26 woody and non-woody fuels and experimental data derived from two combustion test campaigns with an automatically stoked small-scale boiler were investigated through principal component analysis. Thus, the complex interdependencies between the fuel composition and the resulting bottom ash characteristics and the applicability of existing fuel indices were evaluated. The chemometric analysis highlighted that Si, Ca, K, Mg, and also the remaining Al and S in the bottom ash are crucial fuel components in the context of bottom ash melting. On this basis, the molar ratio (Si + P + K)/(Ca + Mg) was adapted and correlated with the susceptibility to slag formation which is a new parameter derived from ash content, slag fraction > 16 mm in the bottom ash, and slag category. Thus, the applicability of a newly developed fuel index was evaluated with respect to the bottom ash slagging risk during real-scale combustion. Three ranges were distinguished for the fuel index corresponding to a specific susceptibility to slag formation (i.e., low 75 mol/g for straw-like fuels and blends with wood). The linear regression of the fuel index with susceptibility to slag formation exhibits a high coefficient of determination (i.e., 0.99 for woody biomass and 0.84 for straw-like fuels and their blends with wood).

Wood chip quality is essential for failure-free and low-emission combustion in automatically stoked small-scale biomass boilers (

Within six case studies, different drying and sieving process steps were employed for the removal of adhering soil and other extraneous impurities from wood chips. Consequently, it was systematically investigated to which extent this strategy can be used to jointly mitigate the risk of bottom ash slagging and high pollutant emission levels during combustion in an automatically stoked small-scale boiler. Throughout all combustion tests, slag and emission formation were recorded. Formation of agglomerates in the bottom ash was not observed in the fuel bed. However, fuel processing resulted in an increase of the ash shrinking–softening range up to 230 K indicating a lower slagging risk in the bottom ash. An asymptotic trend for the ash melting temperatures was observed as a function of the molar (Si + P + K)/(Ca + Mg) ratio. It was also found that potassium is less efficiently retained in the bottom ash with lower Si content in the fuel. Lower moisture contents in the wood chips typically resulted in lower CO emissions and higher boiler efficiencies for the investigated range of moisture content. The sieving of the unprocessed wood chips reduced NOx emission levels up to 28%. However, fuel processing did not necessarily reduce the level of particulate matter emissions.

The reduction in greenhouse gas (GHG) emissions by shifting towards renewable energy sources to control global warming is one of the main challenges of the 21st century [...]

One of the main challenges in biomass combustion for energy and material applications are existing uncertainties for the prediction of slag formation in the bottom ash. Slagging indexes, which are calculated based on the fuel ash composition, can play an important role as simple tools to address this challenge. However, in order to use a fuel index, it is pivotal to consider its relevant scope and its applicability according to its chemical background. In this work, a self-developed Python code was employed to find relevant fuel indexes for the prediction of slag formation with high correlation coefficient based on experimental data from the combustion of rice husk and rice straw. Pre-treatment and fuel blending were utilized to mitigate slag formation in these silica-rich biomass fuels. Results showed that the selected newly defined fuel index, i.e. (K + Na + Mg) / P [mol/mol], which includes ash forming elements relevant for slag formation during the combustion of silica-rich biomass, improves average coefficient of determination for the prediction of the slag formation between 800 and 1100 °C to 0.9187. By employing gas sorption as well as a scanning electron microscopy analysis, it was also shown that the fuel indexes are able to classify the silica-rich ashes for material applications in terms of slag formation on microscopic level and the changes in silica purity. At temperatures higher than 900 °C, porosity of the ashes decreases substantially because of ash sintering on microscopic level. This also applies for acid-leached fuels although their slag formation tendency on macroscopic level is lower than 30 %. These findings are highly relevant to secure sustainable operation of biomass boilers and high-quality biogenic silica production from silica-rich biomass assortments for material applications.

In wood fuels, potassium is by far the most abundant aerosol forming component. Given this predominant role, a major contribution for meeting the tightened total particulate matter emission thresholds on European level could thus be expected by the introduction of an upper limit for potassium content in wood pellets. To evaluate options to introduce such an upper limit for the potassium content of wood pellets, an extensive pellet screening was performed in Germany covering 22 of the 46 ENplus certified wood pellet producers representing more than 50% of the current German wood pellet production of about 2 million t/year. This screening was accompanied by the compilation of wood pellet fuel data from different sources. In total, analysis data of 249 high quality wood pellet samples has been evaluated. Almost all pellet samples originated from European production whereof the majority were pellets from certified German pellet producers. The pellet screening revealed a very large variation in the potassium content of high quality wood pellets ranging from below detection limit to well above 1000 mg/kg d.b. The results also confirmed the predominant role of potassium among the aerosol forming elements potassium, sodium, lead and zinc in wood samples. Furthermore, the screening highlighted that the ash content is not sufficient to evaluate the potassium content of wood pellets. Analysis of selected raw materials from three different pellet producers revealed a strikingly large variation in the potassium content of the raw materials. Consequently, controlled adjustment of the potassium content in wood pellets in the production process would require dedicated mixtures of different raw materials and the limitation of the share of raw materials with particularly high potassium contents. In this way, the potassium content of wood pellets could be limited and this could contribute to the compliance with strict particulate emission thresholds for small scale combustion appliances. Prerequisite for such an approach would be a fast, simple and cheap possibility to determine the potassium content of raw materials for wood pellet production at the production sites and the incorporation of the potassium control in the quality assurance of the pellet production process. Consequently, wood pellets could be produced that facilitate compliance with particulate matter emission threshold without secondary emission reduction measures.

Biomass is an alternative energy resource to fossil fuels because of its potential to reduce greenhouse gas emissions. However, ash-related problems are serious obstacles for this development, especially for the use in combustion plants. Thus, design and operation of biomass boilers require detailed understanding of ash transformation reactions during thermochemical conversion. To evaluate ash transformation in silica-rich biomass fuels, rice husk and rice straw were selected because of their abundance, limited utilization conflicts with the food sector, as well as their potential in both energy and material applications. This paper reveals ash transformation mechanisms relevant for the ash melting behaviour of silica-rich biomass fuels considering chemical and phase composition of the ashes. In this regard, several advanced spectroscopic methods and diffractometry were employed to characterize the materials. The ash transformation reactions and the viscosity were simulated using thermodynamic equilibrium calculations and a slag viscosity modeling toolbox. The results illustrate the impact of impurities on the atomic structure of the silica resulting in an altered ash melting behaviour and viscosity of the silica-rich ashes. Chemical water washing, acid leaching, and blending of rice straw with rice husk strongly influenced the chemical composition of the ashes and improved ash melting behaviour. The analysis also revealed the correlation between the crystalline fraction and the porosity in silica-rich biomass ashes, as well as a crystallinity threshold. These findings are highly relevant for future investigations in boiler designs and production of biogenic silica for material applications.

Non-woody biomass fuels have a great potential to replace fossil fuels and reduce greenhouse gas emissions. At the same time, their application in small scale combustion appliances for heat production is often associated with increased operational problems (e.g. slagging in the bottom ash or deposit formation) as well as elevated gaseous and particulate matter emission levels. To mitigate these problems, scope and limitation of blending raw materials owing critical fuel composition with less problematic biomasses have been systematically studied during combustion experiments in a commercially available small scale combustion appliance with a nominal heat capacity of 30 kW. Three pellet batches of pure biomass (i.e. pine wood, miscanthus and wheat straw) and seven blended biomass pellet batches have been employed. Slag formation in the bottom ash as well as amount of agglomerated bottom ash particles were monitored and evaluated with respect to the determined ash melting temperatures and the measured fuel bed temperatures. Significant reduction of the slagging risk in the bottom ash during combustion of herbaceous fuels can only be achieved for high blending ratios with more than 70 wt% wood. It was furthermore studied to which extent fuel indices are applicable for the prediction of the bottom ash behavior of blended biomass fuels. Furthermore, ternary diagrams were used to rationalize the observed slagging characteristics. It was found that ternary diagrams are superior for a pre-evaluation of the slagging risk in the bottom ash to fuel indices alone. With the knowledge of the fuel ash composition and the ash melting temperatures, a well based pre-evaluation of utilization options can be performed.

Elevated contents of potassium in low quality residues from forestry and agriculture are recognized as main reason for high particulate matter emissions and an increased slagging risk in the bottom ash during utilization of these assortments. Determination of potassium content with reliable rapid and simple test methods could contribute to an appropriate selection and optimum use of biomass for combustion, gasification, and pyrolysis. A cheap soil analysis test kit with investment costs below 1000 € was used to determine potassium contents in different wood assortments. Sample preparation (e.g. particle size) as well as the analysis procedure (e.g. dilution rates, solution agents, and analysis time) were adapted to the requirements for the analysis of the biomass matrix. Activated carbon had to be added to the reagent to improve the precision of the analysis method. The rapid test method is most suitable for potassium contents between 500 and 2000 mg·kg−1 d.b. Potassium contents higher than 2000 mg·kg−1 d.b. can be well detected though with a higher variance. Reliable results were achieved within 10 min analysis time and low cost of about 5 € per sample (consumables). Measurement accuracy was evaluated using ICP-OES analysis of the fuels after digestion with hydrofluoric acid and reveals an overall underestimation of the potassium content though on a comparable level over the whole analysis range.

In Germany, solid biomass fuels based on agricultural byproducts are only used in marginal amounts for small scale combustion. This is the consequence of several regulatory constraints, in particular requirements defined in the first ordinance of the German emission control act (1. BImSchV) including the mandatory utilization of dedicated licensed boilers for such fuels. For the licensing, test fuels with defined fuel composition representing straw and cereal grain like fuels are demanded, and strict emission thresholds have to be met both during type testing and during periodic chimney sweep measurements. To facilitate the market introduction of the first licensed boiler, agricultural biomass test fuels with characteristics being representative for the composition of these assortments were produced and utilized for combustion tests. Emission measurements (i.e., for CO, NOx, PCDD/F, and total particulate matter) were performed by an accredited institute according to the relevant methods. It was demonstrat...

Aspen Plus has become one of the most common process simulation tools for both academia and industrial applications. In the last decade, the number of the papers on Aspen Plus modeling of biomass gasification has significantly increased. This review focuses on recent developments and studies on modeling biomass gasification in Aspen Plus including key aspects such as tar formation and model validation. Accordingly, challenges in modeling due to specific assumptions and limitations will be highlighted to provide a useful basis for researchers and end-users for further process modeling of biomass gasification in Aspen Plus.

Sample tracking and identity are essential when processing multiple samples in parallel. Sequencing applications often involve high sample numbers, and the data are frequently used in a clinical setting. As such, a simple and accurate intrinsic sample tracking process through a sequencing pipeline is essential. Various solutions have been implemented to verify sample identity, including variant detection at the start and end of the pipeline using arrays or genotyping, bioinformatic comparisons, and optical barcoding of samples. None of these approaches are optimal. To establish a more effective approach using genetic barcoding, we developed a panel of unique DNA sequences cloned into a common vector. A unique DNA sequence is added to the sample when it is first received and can be detected by PCR and/or sequencing at any stage of the process. The control sequences are approximately 200 bases long with low identity to any sequence in the National Center for Biotechnology Information nonredundant database (30 bases) and contain no long homopolymer (7) stretches. When a spiked next-generation sequencing library is sequenced, sequence reads derived from this control sequence are generated along with the standard sequencing run and are used to confirm sample identity and determine cross-contamination levels. This approach is used in our targeted clinical diagnostic whole-genome and RNA-sequencing pipelines and is an inexpensive, flexible, and platform-agnostic solution.

Non-woody biomass fuels have a great potential to replace fossil fuels and reduce greenhouse gas emissions. At the same time, their application in small scale combustion appliances for heat production is often associated with increased operational problems (e.g. slagging in the bottom ash or fouling) as well as elevated particulate and gaseous emission levels. To mitigate these problems, scope and limitation of blending raw materials owing critical fuel composition with less problematic biomasses have been systematically studied during combustion experiments in a commercially available small scale combustion appliance with a nominal heat capacity of 30 kW. Three pellet batches of pure biomass (i.e. pine wood, miscanthus and wheat straw) as well as seven blended biomass pellet batches have been employed. Slagging, emission of total particulate matter (TPM) and gaseous emissions (i.e. CO, NOx, SO2 and HCl) were monitored. The results were evaluated with respect to the emission reduction potential of the blending strategy as well as the applicability of fuel indices which were originally developed for coal and pure biomass fuels. Based on the results, blending of herbaceous raw materials with woody biomass reduces the slagging risk in the bottom ash and leads to reduced emission levels, though significant reduction potential was observed only for blends with at least 50 wt% wood. The blending of miscanthus with wood seemed to be more effective. Most of the fuel indices which were deduced from the chemical composition of the fuel seem to be applicable for a preliminary evaluation of blended biomass fuels and the prediction of critical emission levels.

Context and Objective:Oncocytic thyroid carcinoma, also known as Hürthle cell thyroid carcinoma, accounts for only a small percentage of all thyroid cancers. However, this malignancy often presents at an advanced stage and poses unique challenges to patients and clinicians. Surgical resection of the tumor accompanied in some cases by radioactive iodine treatment, radiation, and chemotherapy are the established modes of therapy. Knowledge of the perturbed oncogenic pathways can provide better understanding of the mechanism of disease and thus opportunities for more effective clinical management.Design and Patients:Initially, two oncocytic thyroid carcinomas and their matched normal tissues were profiled using whole genome sequencing. Subsequently, 72 oncocytic thyroid carcinomas, one cell line, and five Hürthle cell adenomas were examined by targeted sequencing for the presence of mutations in the multiple endocrine neoplasia I (MEN1) gene.Results:Here we report the identification of MEN1 loss-of-function mutations in 4% of patients diagnosed with oncocytic thyroid carcinoma. Whole genome sequence data also revealed large regions of copy number variation encompassing nearly the entire genomes of these tumors.Conclusion:Menin, a ubiquitously expressed nuclear protein, is a well-characterized tumor suppressor whose loss is the cause of MEN1 syndrome. Menin is involved in several major cellular pathways such as regulation of transcription, control of cell cycle, apoptosis, and DNA damage repair pathways. Mutations of this gene in a subset of Hürthle cell tumors point to a potential role for this protein and its associated pathways in thyroid tumorigenesis.

SummaryHigh-grade serous ovarian cancer exhibits extensive intratumoral heterogeneity coupled with widespread intraperitoneal disease. Despite this, metastatic spread of tumor clones is non-random, implying the existence of local microenvironmental factors that shape tumor progression. We interrogated the molecular interface between tumor-infiltrating lymphocytes (TIL) and cancer cells in 143 samples from 21 patients using whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T- and B-cell receptor sequencing. We identify 3 immunologic response categories, which frequently co-exist within individual patients. Furthermore, epithelial CD8+ TIL were inversely associated with malignant cell diversity, evidenced by subclonal neoepitope elimination and spatial tracking between tumor and T-cell clones. Intersecting mutational signatures and immune analysis showed that foldback inversion genomic aberrations lead to worse outcomes even in the presence of cytotoxic TIL (n=433). Thus, regional variation in immune contexture mirrors the pattern of intraperitoneal malignant spread, provoking new perspectives for treatment of this challenging disease.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

SummaryCellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.

Utilization of biomass either as a renewable energy source or for the generation of biogenic materials has received considerable interest during the past years. In the case of rice husk (RH) and rice straw (RS) with high silica contents in the fuel ash, these approaches can be combined to produce high-grade biogenic silica with purities >98 wt % from combustion residues. The overall process can be considered nearly neutral in terms of CO2 emission and global warming, but it can also address disposal challenges of rice husk and rice straw. For the resulting biogenic silica, several advanced application opportunities exist, e.g., as adsorbents, catalysts, drug delivery systems, etc. This article provides a comprehensive literature review on rice husk and rice straw combustion as well as applied strategies for raw material pre-treatment and/or post-treatment of resulting ashes to obtain high quality biogenic silica. Purity of up to 97.2 wt % SiO2 can be reached by combustion of untreated material. With appropriate fuel pre-treatment and ash post-treatment, biogenic silica with purity up to 99.7 wt % can be achieved. Studies were performed almost exclusively at a laboratory scale.

High-grade serous carcinoma (HGSC) is the most common and fatal form of ovarian cancer. While most tumours are highly sensitive to cytoreductive surgery and platinum- and taxane-based chemotherapy, the majority of patients experience recurrence of treatment-resistant tumours. The clonal origin and mutational adaptations associated with recurrent disease are poorly understood. We performed whole exome sequencing on tumour cells harvested from ascites at three time points (primary, first recurrence, and second recurrence) for three HGSC patients receiving standard treatment. Somatic point mutations and small insertions and deletions were identified by comparison to constitutional DNA. The clonal structure and evolution of tumours were inferred from patterns of mutant allele frequencies. TP53 mutations were predominant in all patients at all time points, consistent with the known founder role of this gene. Tumours from all three patients also harboured mutations associated with cell cycle checkpoint function and Golgi vesicle trafficking. There was convergence of germline and somatic variants within the DNA repair, ECM, cell cycle control, and Golgi vesicle pathways. The vast majority of somatic variants found in recurrent tumours were present in primary tumours. Our findings highlight both known and novel pathways that are commonly mutated in HGSC. Moreover, they provide the first evidence at single nucleotide resolution that recurrent HGSC arises from multiple clones present in the primary tumour with negligible accumulation of new mutations during standard treatment.

The classification of endometrial carcinomas is based on pathological assessment of tumour cell type; the different cell types (endometrioid, serous, carcinosarcoma, mixed, undifferentiated, and clear cell) are associated with distinct molecular alterations. This current classification system for high-grade subtypes, in particular the distinction between high-grade endometrioid (EEC-3) and serous carcinomas (ESC), is limited in its reproducibility and prognostic abilities. Therefore, a search for specific molecular classifiers to improve endometrial carcinoma subclassification is warranted. We performed target enrichment sequencing on 393 endometrial carcinomas from two large cohorts, sequencing exons from the following nine genes: ARID1A, PPP2R1A, PTEN, PIK3CA, KRAS, CTNNB1, TP53, BRAF, and PPP2R5C. Based on this gene panel, each endometrial carcinoma subtype shows a distinct mutation profile. EEC-3s have significantly different frequencies of PTEN and TP53 mutations when compared to low-grade endometrioid carcinomas. ESCs and EEC-3s are distinct subtypes with significantly different frequencies of mutations in PTEN, ARID1A, PPP2R1A, TP53, and CTNNB1. From the mutation profiles, we were able to identify subtype outliers, ie cases diagnosed morphologically as one subtype but with a mutation profile suggestive of a different subtype. Careful review of these diagnostically challenging cases suggested that the original morphological classification was incorrect in most instances. The molecular profile of carcinosarcomas suggests two distinct mutation profiles for these tumours: endometrioid-type (PTEN, PIK3CA, ARID1A, KRAS mutations) and serous-type (TP53 and PPP2R1A mutations). While this nine-gene panel does not allow for a purely molecularly based classification of endometrial carcinoma, it may prove useful as an adjunct to morphological classification and serve as an aid in the classification of problematic cases. If used in practice, it may lead to improved diagnostic reproducibility and may also serve to stratify patients for targeted therapeutics. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The clustered homeobox proteins play crucial roles in development, hematopoiesis, and leukemia, yet the targets they regulate and their mechanisms of action are poorly understood. Here, we identified the binding sites for Hoxa9 and the Hox cofactor Meis1 on a genome-wide level and profiled their associated epigenetic modifications and transcriptional targets. Hoxa9 and the Hox cofactor Meis1 cobind at hundreds of highly evolutionarily conserved sites, most of which are distant from transcription start sites. These sites show high levels of histone H3K4 monomethylation and CBP/P300 binding characteristic of enhancers. Furthermore, a subset of these sites shows enhancer activity in transient transfection assays. Many Hoxa9 and Meis1 binding sites are also bound by PU.1 and other lineage-restricted transcription factors previously implicated in establishment of myeloid enhancers. Conditional Hoxa9 activation is associated with CBP/P300 recruitment, histone acetylation, and transcriptional activation of a network of proto-oncogenes, including Erg, Flt3, Lmo2, Myb, and Sox4. Collectively, this work suggests that Hoxa9 regulates transcription by interacting with enhancers of genes important for hematopoiesis and leukemia.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Ovarian clear-cell and endometrioid carcinomas may arise from endometriosis, but the molecular events involved in this transformation have not been described.We sequenced the whole transcriptomes of 18 ovarian clear-cell carcinomas and 1 ovarian clear-cell carcinoma cell line and found somatic mutations in ARID1A (the AT-rich interactive domain 1A [SWI-like] gene) in 6 of the samples. ARID1A encodes BAF250a, a key component of the SWI–SNF chromatin remodeling complex. We sequenced ARID1A in an additional 210 ovarian carcinomas and a second ovarian clear-cell carcinoma cell line and measured BAF250a expression by means of immunohistochemical analysis in an additional 455 ovarian carcinomas.ARID1A mutations were seen in 55 of 119 ovarian clear-cell carcinomas (46%), 10 of 33 endometrioid carcinomas (30%), and none of the 76 high-grade serous ovarian carcinomas. Seventeen carcinomas had two somatic mutations each. Loss of the BAF250a protein correlated strongly with the ovarian clear-cell carcinoma and endometrioid carcinoma subtypes and the presence of ARID1A mutations. In two patients, ARID1A mutations and loss of BAF250a expression were evident in the tumor and contiguous atypical endometriosis but not in distant endometriotic lesions.These data implicate ARID1A as a tumor-suppressor gene frequently disrupted in ovarian clear-cell and endometrioid carcinomas. Since ARID1A mutation and loss of BAF250a can be seen in the preneoplastic lesions, we speculate that this is an early event in the transformation of endometriosis into cancer. (Funded by the British Columbia Cancer Foundation and the Vancouver General Hospital–University of British Columbia Hospital Foundation.).

In alternative expression analysis by sequencing (ALEXA-seq), we developed a method to analyze massively parallel RNA sequence data to catalog transcripts and assess differential and alternative expression of known and predicted mRNA isoforms in cells and tissues. As proof of principle, we used the approach to compare fluorouracil-resistant and -nonresistant human colorectal cancer cell lines. We assessed the sensitivity and specificity of the approach by comparison to exon tiling and splicing microarrays and validated the results with reverse transcription-PCR, quantitative PCR and Sanger sequencing. We observed global disruption of splicing in fluorouracil-resistant cells characterized by expression of new mRNA isoforms resulting from exon skipping, alternative splice site usage and intron retention. Alternative expression annotation databases, source code, a data viewer and other resources to facilitate analysis are available at http://www.alexaplatform.org/alexa_seq/.

We describe a new method, Tag-seq, which employs ultra high-throughput sequencing of 21 base pair cDNA tags for sensitive and cost-effective gene expression profiling. We compared Tag-seq data to LongSAGE data and observed improved representation of several classes of rare transcripts, including transcription factors, antisense transcripts, and intronic sequences, the latter possibly representing novel exons or genes. We observed increases in the diversity, abundance, and dynamic range of such rare transcripts and took advantage of the greater dynamic range of expression to identify, in cancers and normal libraries, altered expression ratios of alternative transcript isoforms. The strand-specific information of Tag-seq reads further allowed us to detect altered expression ratios of sense and antisense (S-AS) transcripts between cancer and normal libraries. S-AS transcripts were enriched in known cancer genes, while transcript isoforms were enriched in miRNA targeting sites. We found that transcript abundance had a stronger GC-bias in LongSAGE than Tag-seq, such that AT-rich tags were less abundant than GC-rich tags in LongSAGE. Tag-seq also performed better in gene discovery, identifying >98% of genes detected by LongSAGE and profiling a distinct subset of the transcriptome characterized by AT-rich genes, which was expressed at levels below those detectable by LongSAGE. Overall, Tag-seq is sensitive to rare transcripts, has less sequence composition bias relative to LongSAGE, and allows differential expression analysis for a greater range of transcripts, including transcripts encoding important regulatory molecules.

We characterized the relationship of H3K4me1 and H3K4me3 at distal and proximal regulatory elements by comparing ChIP-seq profiles for these histone modifications and for two functionally different transcription factors: STAT1 in the immortalized HeLa S3 cell line, with and without interferon-gamma (IFNG) stimulation; and FOXA2 in mouse adult liver tissue. In unstimulated and stimulated HeLa cells, respectively, we determined approximately 270,000 and approximately 301,000 H3K4me1-enriched regions, and approximately 54,500 and approximately 76,100 H3K4me3-enriched regions. In mouse adult liver, we determined approximately 227,000 and approximately 34,800 H3K4me1 and H3K4me3 regions. Seventy-five percent of the approximately 70,300 STAT1 binding sites in stimulated HeLa cells and 87% of the approximately 11,000 FOXA2 sites in mouse liver were distal to known gene TSS; in both cell types, approximately 83% of these distal sites were associated with at least one of the two histone modifications, and H3K4me1 was associated with over 96% of marked distal sites. After filtering against predicted transcription start sites, 50% of approximately 26,800 marked distal IFNG-stimulated STAT1 binding sites, but 95% of approximately 5800 marked distal FOXA2 sites, were associated with H3K4me1 only. Results for HeLa cells generated additional insights into transcriptional regulation involving STAT1. STAT1 binding was associated with 25% of all H3K4me1 regions in stimulated HeLa cells, suggesting that a single transcription factor can interact with an unexpectedly large fraction of regulatory regions. Strikingly, for a large majority of the locations of stimulated STAT1 binding, the dominant H3K4me1/me3 combinations were established before activation, suggesting mechanisms independent of IFNG stimulation and high-affinity STAT1 binding.

There is substantial heterogeneity among primary prostate cancers, evident in the spectrum of molecular abnormalities and its variable clinical course. As part of The Cancer Genome Atlas (TCGA), we present a comprehensive molecular analysis of 333 primary prostate carcinomas. Our results revealed a molecular taxonomy in which 74% of these tumors fell into one of seven subtypes defined by specific gene fusions (ERG, ETV1/4, and FLI1) or mutations (SPOP, FOXA1, and IDH1). Epigenetic profiles showed substantial heterogeneity, including an IDH1 mutant subset with a methylator phenotype. Androgen receptor (AR) activity varied widely and in a subtype-specific manner, with SPOP and FOXA1 mutant tumors having the highest levels of AR-induced transcripts. 25% of the prostate cancers had a presumed actionable lesion in the PI3K or MAPK signaling pathways, and DNA repair genes were inactivated in 19%. Our analysis reveals molecular heterogeneity among primary prostate cancers, as well as potentially actionable molecular defects., National Institutes of Health (U.S.). (grant 5U24CA143799), National Institutes of Health (U.S.). (grant 5U24CA143835), National Institutes of Health (U.S.). (grant 5U24CA143840), National Institutes of Health (U.S.). (grant 5U24CA143843), National Institutes of Health (U.S.). (grant 5U24CA143845), National Institutes of Health (U.S.). (grant 5U24CA143848), National Institutes of Health (U.S.). (grant 5U24CA143858), National Institutes of Health (U.S.). (grant 5U24CA143866), National Institutes of Health (U.S.). (grant 5U24CA143867), National Institutes of Health (U.S.). (grant 5U24CA143882), National Institutes of Health (U.S.). (grant 5U24CA143883), National Institutes of Health (U.S.). (grant 5U24CA144025), National Institutes of Health (U.S.). (grant U54HG003067), National Institutes of Health (U.S.). (grant U54HG003079), National Institutes of Health (U.S.). (grant U54HG003273), National Institutes of Health (U.S.). (grant P30CA16672)

The development of targeted anti-cancer therapies through the study of cancer genomes is intended to increase survival rates and decrease treatment-related toxicity. We treated a transposon-driven, functional genomic mouse model of medulloblastoma with 'humanized' in vivo therapy (microneurosurgical tumour resection followed by multi-fractionated, image-guided radiotherapy). Genetic events in recurrent murine medulloblastoma exhibit a very poor overlap with those in matched murine diagnostic samples (

Genome sequences of chicken (low pathogenic avian influenza [LPAI] and highly pathogenic avian influenza [HPAI]) and human isolates from a 2004 outbreak of H7N3 avian influenza in Canada showed a novel insertion in the HA0 cleavage site of the human and HPAI isolate. This insertion likely occurred by recombination between the hemagglutination and matrix genes in the LPAI virus.

The Mammalian Gene Collection (MGC) consortium (http://mgc.nci.nih.gov) seeks to establish publicly available collections of full-ORF cDNAs for several organisms of significance to biomedical research, including human. To date over 15,200 human cDNA clones containing full-length open reading frames (ORFs) have been identified via systematic expressed sequence tag (EST) analysis of a diverse set of cDNA libraries; however, further systematic EST analysis is no longer an efficient method for identifying new cDNAs. As part of our involvement in the MGC program, we have developed a scalable method for targeted recovery of cDNA clones to facilitate recovery of genes absent from the MGC collection. First, cDNA is synthesized from various RNAs, followed by polymerase chain reaction (PCR) amplification of transcripts in 96-well plates using gene-specific primer pairs flanking the ORFs. Amplicons are cloned into a sequencing vector, and full-length sequences are obtained. Sequences are processed and assembled using Phred and Phrap, and analyzed using Consed and a number of bioinformatics methods we have developed. Sequences are compared with the Reference Sequence (RefSeq) database, and validation of sequence discrepancies is attempted using other sequence databases including dbEST and dbSNP. Clones with identical sequence to RefSeq or containing only validated changes will become part of the MGC human gene collection. Clones containing novel splice variants or polymorphisms have also been identified. Our approach to clone recovery, applied at large scale, has the potential to recover many and possibly most of the genes absent from the MGC collection.

High-grade serous ovarian cancer (HGSC) is characterized by poor outcome, often attributed to the emergence of treatment-resistant subclones. We sought to measure the degree of genomic diversity within primary, untreated HGSCs to examine the natural state of tumour evolution prior to therapy. We performed exome sequencing, copy number analysis, targeted amplicon deep sequencing and gene expression profiling on 31 spatially and temporally separated HGSC tumour specimens (six patients), including ovarian masses, distant metastases and fallopian tube lesions. We found widespread intratumoural variation in mutation, copy number and gene expression profiles, with key driver alterations in genes present in only a subset of samples (eg PIK3CA, CTNNB1, NF1). On average, only 51.5% of mutations were present in every sample of a given case (range 10.2-91.4%), with TP53 as the only somatic mutation consistently present in all samples. Complex segmental aneuploidies, such as whole-genome doubling, were present in a subset of samples from the same individual, with divergent copy number changes segregating independently of point mutation acquisition. Reconstruction of evolutionary histories showed one patient with mixed HGSC and endometrioid histology, with common aetiologic origin in the fallopian tube and subsequent selection of different driver mutations in the histologically distinct samples. In this patient, we observed mixed cell populations in the early fallopian tube lesion, indicating that diversity arises at early stages of tumourigenesis. Our results revealed that HGSCs exhibit highly individual evolutionary trajectories and diverse genomic tapestries prior to therapy, exposing an essential biological characteristic to inform future design of personalized therapeutic solutions and investigation of drug-resistance mechanisms.