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Starting with SAGE-libraries prepared from C. elegans FAC-sorted embryonic intestine cells (8E–16E cell stage), from total embryos and from purified oocytes, and taking advantage of the NextDB in situ hybridization data base, we define sets of genes highly expressed from the zygotic genome, and expressed either exclusively or preferentially in the embryonic intestine or in the intestine of newly hatched larvae; we had previously defined a similarly expressed set of genes from the adult intestine. We show that an extended TGATAA-like sequence is essentially the only candidate for a cis-acting regulatory motif common to intestine genes expressed at all stages. This sequence is a strong ELT-2 binding site and matches the sequence of GATA-like sites found to be important for the expression of every intestinal gene so far analyzed experimentally. We show that the majority of these three sets of highly expressed intestinal-specific/intestinal-enriched genes respond strongly to ectopic expression of ELT-2 within the embryo. By flow-sorting elt-2(null) larvae from elt-2(+) larvae and then preparing Solexa/Illumina-SAGE libraries, we show that the majority of these genes also respond strongly to loss-of-function of ELT-2. To test the consequences of loss of other transcription factors identified in the embryonic intestine, we develop a strain of worms that is RNAi-sensitive only in the intestine; however, we are unable (with one possible exception) to identify any other transcription factor whose intestinal loss-of-function causes a phenotype of comparable severity to the phenotype caused by loss of ELT-2. Overall, our results support a model in which ELT-2 is the predominant transcription factor in the post-specification C. elegans intestine and participates directly in the transcriptional regulation of the majority (>80%) of intestinal genes. We present evidence that ELT-2 plays a central role in most aspects of C. elegans intestinal physiology: establishing the structure of the enterocyte, regulating enzymes and transporters involved in digestion and nutrition, responding to environmental toxins and pathogenic infections, and regulating the downstream intestinal components of the daf-2/daf-16 pathway influencing aging and longevity.

CK syndrome (CKS) is an X-linked recessive intellectual disability syndrome characterized by dysmorphism, cortical brain malformations, and an asthenic build. Through an X chromosome single-nucleotide variant scan in the first reported family, we identified linkage to a 5 Mb region on Xq28. Sequencing of this region detected a segregating 3 bp deletion (c.696_698del [p.Lys232del]) in exon 7 of NAD(P) dependent steroid dehydrogenase-like (NSDHL), a gene that encodes an enzyme in the cholesterol biosynthesis pathway. We also found that males with intellectual disability in another reported family with an NSDHL mutation (c.1098 dup [p.Arg367SerfsX33]) have CKS. These two mutations, which alter protein folding, show temperature-sensitive protein stability and complementation in Erg26-deficient yeast. As described for the allelic disorder CHILD syndrome, cells and cerebrospinal fluid from CKS patients have increased methyl sterol levels. We hypothesize that methyl sterol accumulation, not only cholesterol deficiency, causes CKS, given that cerebrospinal fluid cholesterol, plasma cholesterol, and plasma 24S-hydroxycholesterol levels are normal in males with CKS. In summary, CKS expands the spectrum of cholesterol-related disorders and insight into the role of cholesterol in human development.

This paper investigates the problem of target position estimation with a single-observer passive coherent location (PCL) system. An approach that combines angle with time difference of arrival (ATDOA) is used to estimate the location of a target. Compared with the TDOA-only method which needs two steps, the proposed method estimates the target position more directly. The constrained total least squares (CTLS) technique is applied in this approach. It achieves the Cramer–Rao lower bound (CRLB) when the parameter measurements are subject to small Gaussian-distributed errors. Performance analysis and the CRLB of this approach are also studied. Theory verifies that the ATDOA method gets a lower CRLB than the TDOA-only method with the same TDOA measuring error. It can also be seen that the position of the target affects estimating precision. At the same time, the locations of transmitters affect the precision and its gradient direction. Compared with the TDOA, the ATDOA method can obtain more precise target position estimation. Furthermore, the proposed method accomplishes target position estimation with a single transmitter, while the TDOA-only method needs at least four transmitters to get the target position. Furthermore, the transmitters’ position errors also affect precision of estimation regularly.

Abstract 439 Introduction: Follicular lymphoma (FL) is the most common indolent lymphoid malignancy in North America with approximately 20,000 new cases of this incurable cancer diagnosed each year. In approximately 85% of patients, FL is associated with the reciprocal translocation t(14;18)(q32;q21), which results in a fusion between IGH and BCL2 genes and consequent over-expression of the anti-apoptotic protein BCL2. This translocation likely represents an initiating event for FL, requiring additional mutational events for the onset of clinical disease. To investigate the relationship between genome rearrangements and FL we identified rearrangement locations in the genome followed by detailed, fine-structure analysis of the rearrangements to ascertain their effects on genes and other features of biological interest. Patients and Methods: We used a whole-genome bacterial artificial chromosome (BAC) fingerprint-based approach, termed Fingerprint Profiling (FPP, Krzywinski, M. et al. 2007), to detect genome rearrangements relative to the reference human genome in neoplastic B cells purified from 24 FL patient biopsies. Analysis of 2,640,707 BAC fingerprints revealed 721 candidate genomic rearrangements. To validate these observations and provide base-pair resolution of the rearrangement breakpoints we performed paired-end massively parallel sequencing, on the Illumina Genome Analyzer II platform, of the breakpoint-containing regions captured in the BAC clones. Sequence reads were assembled into contigs using our in-house de novo assembly algorithm ABySS (Assembly By Short Sequences, Simpson, J. et al. 2009) then aligned to the reference human genome. Following manual annotation of the breakpoint junctions PCR primers were designed to assay patient tumour and matched constitutional DNA and thus determine whether the observed genome rearrangements were somatic (acquired) or germline in origin. Results: 727 BACs with apparent large-scale genome rearrangements, representing 354 distinct genome rearrangements across 20 patients, were sequenced in 95 pools, generating 72 Gbp of sequence. The 354 distinct events include 163 deletions, 71 inversions, 27 insertions, 83 translocations and 10 duplications, ranging in size from 3 kb to 67 Mb. PCR assays for 194 of the distinct events have been performed thus far identifying 80 distinct somatic and 114 germline-derived structural variations at base-pair resolution. Of the somatic events 5 are present in two or more of the 20 patients analyzed including a 720 kb inversion of 3q27.3 that results in expression of a BCL6-ST6GAL1 fusion transcript. Identification at base-pair resolution of breakpoint sequences enabled a detailed study of breakpoint and fusion mechanisms. We classified breakpoint junctions into 4 groups; those with microhomology (48%), those with sequence additions (28%), those with blunt fusions (20%) and those with flanking low copy repeats (4%). We were particularly interested in establishing the origin of the observed nucleotide sequence additions in 97 breakpoint junctions. The sequence additions ranged in size from a single nucleotide to 454 bp. In one case we have unambiguously mapped a 53 bp sequence, lying within one of the 3q27.3 inversion breakpoints, to chromosome 5q12.3. This finding is consistent with the recently proposed fork stalling and template switching (FoSTeS) DNA replication-based mechanism and thus represents a novel mechanism in FL lymphomagenesis. Conclusions: We have successfully employed high-throughput clone fingerprinting and sequencing to identify numerous novel somatic and germline genome rearrangements from FL primary tumour samples. Furthermore, base-pair resolution of rearrangement breakpoints provides mechanistic insights. With the complete inventory of somatic and germline events in hand we will be able to propose recurrent structurally altered genes in FL patients for validation in independent datasets and improve our understanding of FL biology. Pathway analyses to identify emerging themes from somatic mutations are also being performed. The PCR assays we have developed will also be of utility in identifying germline predisposition alleles in larger FL patient cohorts. Disclosures: No relevant conflicts of interest to declare.

Abstract 139 Background: Diffuse large B cell lymphoma (DLBCL) and follicular lymphoma (FL) constitute 70% of all non-Hodgkin lymphomas (NHL). Both malignancies derive from germinal center (GC) B-cells and are characterized by clinical and genomic heterogeneity. Chromosomal alterations that deregulate oncogenes as well as mutations in genes involved in cell proliferation and apoptosis have been described in DLBCL. This disease can also be divided into distinct molecular subtypes by gene expression profiling. Differences observed between the activated B-cell (ABC) and germinal center B cell (GCB) subtypes result in part from distinct genomic alterations (Lenz, PNAS 2008) . For example, recent targeted re-sequencing efforts have revealed mutations in various genes in the NFkB signaling pathway, which likely contribute mainly to the ABC subtype (Compagno, Nature 2009). Thus far, few GCB-specific mutations other than t(14;18) have been identified. Methods: Targeted re-sequencing studies are only able to reveal mutations in pre-selected candidate genes and so much of the genome has not yet been investigated in lymphomas. To obtain a more global view of the mutational landscape of NHL, we applied Illumina massively parallel second-generation sequencing to sequence the genomic DNA of a FL tumor sample. In parallel, we sequenced the transcriptomes (i.e. mRNA) of 31 DLBCL tumor samples using the same technology. Results: Genomic sequencing revealed a mutation altering tyrosine 641 (Y641) in the polycomb group oncogene Enhancer of Zeste Homolog 2 (EZH2), a gene responsible for trimethylating lysine 27 on histone H3 (H3K27). Mutations affecting the same codon were also observed in 4 DLBCL samples. These mutations were confirmed to be somatic in nature by Sanger sequencing exon 15 in tumor DNA and germline DNA derived from peripheral blood in these 5 patients. Sanger sequencing all coding exons in EZH2 from an additional 25 FL samples revealed that mutations were restricted to codon 641. These mutations could change the tyrosine residue to Asparagine, Serine, Phenylalanine or Histidine. The frequency of EZH2 Y641 mutations in GCB-derived lymphomas was determined by Sanger sequencing exon 15 in a total of 479 lymphoma samples. EZH2 (Y641) mutations occurred predominantly in lymphomas of the GCB type: 18/80 (22%) GCB-type de novo DLBCL, 6/52 (11.5)% of “transformed” DLBCL derived from FL and 16/225 (7.1%) of pre-treatment FL samples. No EZH2 mutations were found in 42 DLBCL of the ABC subype, 25 mantle cell lymphomas, 30 small lymphocytic lymphomas or 25 T cell lymphomas or 23 reactive tonsils. Introducing each of the four Y641 mutations into wild-type EZH2 resulted in 15-fold reduction in the ability to trimethylate lysine 27 on H3 peptides in-vitro. Discussion: Over-expression of EZH2 is thought to drive malignancy in a variety of solid tumors but mutations in this gene have never been described. The trimethylated H3K27 epigenetic mark is used to silence the transcription of genes involved in differentiation and hematopoiesis. In Drosophila, the phenotypic consequence of mutating the orthologous tyrosine residue in the E(z)(“Enhancer of Zeste”) gene indicates an apparent gain-of-function (Jones, Genetics 1990). Taken together, our mutation frequency and biochemical data are compatible with the notion that alteration of the ability of germinal center B-cells to trimethylate H3K27 may promote the development of FL and the GCB subtype of DLBCL. Disclosures: Zhu: BPS Biosciences: Employment. Kimbara:BPS Biosciences: Employment. Shashkin:BPS Biosciences: Employment. Charlot:BPS Biosciences: Employment. Tcherpakov:BPS Biosciences: Employment.

Abstract 804 Introduction: Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) are the two most common types of non Hodgkin lymphoma (NHL). It is widely accepted that DLBCL can be divided into two major subtypes using gene expression profiling: germinal center B-cell (GCB) and activated B-cell (ABC). Both FL and the GCB subtype of DLBCL derive from germinal center B cells and have been found to share some common mutational events such as translocations leading to the deregulation of the BCL2 oncogene and mutations affecting a single tyrosine (Y641) in the histone methyltransferase EZH2. In contrast, ABC DLBCL tumors are characterized by mutations leading to the constitutive activity of NFkB. The clear differences in treatment response between subtypes allow this distinction to be used as a prognostic indicator and may ultimately lead to therapies that target individual features of each subtype. However, besides the gene expression and mutational signatures that differentiate the DLBCL subtypes, there is a paucity of molecular prognostic markers in these NHLs. Further, there is limited knowledge about the genetic events that drive the GCB subtype of DLBCL, which, if better understood, may enable the design of targeted therapeutics. Methods: To identify mutations driving lymphomagenesis and in particular, aggressive cases of NHL, we applied Illumina second-generation sequencing technology to the analysis of tumor genomes and constitutional DNAs from a FL and a DLBCL tumor and the exomes from two additional DLBCLs. In these “omes”, we identified somatic protein-altering point mutations in more than 250 genes including genes known to be involved in cancer, for example TP53, FAS and TNFAIP3 (A20). Many of these mutations may represent passenger rather than driver mutations, the latter of which are involved in disease progression. To identify the likely driver mutations, we sought to identify the genes that are recurrent targets of somatic mutation in these cancers. To this end, we further analyzed the transcriptome sequences we generated using RNA-seq from 95 primary DLBCLs,13 FL cases and 10 DLBCL-derived cell lines. Results: 105 of the genes found mutated in the FL and DLBCL genomes were observed to be recurrent targets of somatic mutation in these diseases. Some of these were known targets of aberrant somatic hypermutation (SHM) including BCL2, PIM1, and IRF4 and others have been previously identified as targets of recurrent mutation in lymphoma, such as EZH2, CD79B and CARD11. One of the most frequently mutated genes was MLL2, a histone methyltransferase never before implicated in lymphomagenesis. MLL2 showed a pattern of mutation characteristic of a dosage-sensitive tumor suppressor gene. Another frequently mutated gene was MEF2B, a calcium-regulated transcriptional co-activator/repressor that cooperates with histone modifying enzymes to epigenetically regulate the expression of genes. We found that mutations affecting MEF2B occur in 11.7% of FL and 9% of DLBCL, with the majority (73%) of these mutations affecting three amino acids (K4, Y69, and D83). Analysis of these 105 recurrently mutated genes for prognostic signatures is ongoing. Conclusions: High-throughput sequencing platforms have enabled the identification of recurrent targets of somatic mutations never suspected to be involved in lymphoma. Some of these mutated genes may have prognostic value while others may represent targets for the rational design of novel therapeutics. Disclosures: No relevant conflicts of interest to declare.

We analysed primary breast cancers by genomic DNA copy number arrays, DNA methylation, exome sequencing, messenger RNA arrays, microRNA sequencing and reverse-phase protein arrays. Our ability to integrate information across platforms provided key insights into previously defined gene expression subtypes and demonstrated the existence of four main breast cancer classes when combining data from five platforms, each of which shows significant molecular heterogeneity. Somatic mutations in only three genes (TP53, PIK3CA and GATA3) occurred at >10% incidence across all breast cancers; however, there were numerous subtype-associated and novel gene mutations including the enrichment of specific mutations in GATA3, PIK3CA and MAP3K1 with the luminal A subtype. We identified two novel protein-expression-defined subgroups, possibly produced by stromal/microenvironmental elements, and integrated analyses identified specific signalling pathways dominant in each molecular subtype including a HER2/phosphorylated HER2/EGFR/phosphorylated EGFR signature within the HER2-enriched expression subtype. Comparison of basal-like breast tumours with high-grade serous ovarian tumours showed many molecular commonalities, indicating a related aetiology and similar therapeutic opportunities. The biological finding of the four main breast cancer subtypes caused by different subsets of genetic and epigenetic abnormalities raises the hypothesis that much of the clinically observable plasticity and heterogeneity occurs within, and not across, these major biological subtypes of breast cancer.

Abstract 473 Introduction : Follicular lymphoma (FL) and diffuse large B cell lymphoma (DLBCL) are the most common types of B-cell derived non-Hodgkin lymphomas (NHL). A significant proportion of patients with FL develop transformed disease reminiscent of DLBCL, which arises from the cells of FL. A common feature of FL and a subset of DLBCL is the presence of a balanced cytogenetic translocation t(14;18)(q32;q21). In addition to chromosome rearrangements, inappropriate somatic hypermutation of proto-oncogenes, including MYC, PIM1, ARHH and PAX5, have been proposed to contribute to DLBCL, but not to FL. Evidence for other mutations contributing to the development of DLBCL and FL is lacking. Our research group had previously detected a residue of the SET domain of the methyltransferase EZH2 (Y641) that is frequently mutated both in FL and the GCB subtype of DLBCL (Morin, R et al. 2010 Nature Genetics 42 (2):181-5). It has also been recently reported that specific gene expression signatures can reveal unique diagnostic and prognostic information about FL and DLBCL. The presence of mutations in genes that contribute to chromatin modification (from transcriptionally active to silent, and vice versa) can have an effect on the regulation of gene expression, playing an important role in cancer. Methods: We have deep-sequenced the whole transcriptome of DLBCL samples using Illumina second-generation sequencing to detect candidate mutations in different genes that may contribute to lymphoma development and progression. Based on our observations of these data, we subsequently PCR amplified and sequenced the entire MLL2 locus from genomic DNA isolated from FL and DLBCL tumor samples, and from matched germline DNA where available. Using an alternative approach, consisting of a targeted hybridization capture of MEF2B exons from genomic DNA, we re-sequenced the exons of MEF2B in an extended cohort of FL and DLBCL samples. Results: Among many other mutated genes we have characterized the mutations found in two chromatin modifying genes, MLL2 and MEF2B. MLL2, a gene that encodes a histone methyltransferase, is somatically mutated in 89% of FL (n=35) and 32% of DLBCL (n=37) samples. The majority of these mutations were truncations and frame-shift insertions and deletions, likely inactivating MLL2. MEF2B, which encodes a MADS/MEF2 DNA binding protein involved in the regulation of gene expression, was found mutated in 12% of FL (n=274) and 9% of DLBCL (n=321) tumor samples. In contrast with the mutational pattern of MLL2, we did not detect non-sense mutations in MEF2B. All mutations in MEF2B affected a small number of residues. Several studies in MEF2 family members have shown the importance of some of these mutated residues for the correct function of the protein. Conclusions: After high-throughput sequencing of transcriptomes in a cohort of FL and DLBCL lymphoma samples, two targeted second generation re-sequencing approaches have enabled the screening of individual genes in a large cohort of samples. We chose two of the genes with strong evidence for recurrent somatic mutations and no previously known role in lymphoma, MLL2 and MEF2B, for detailed characterization. The high incidence of mutations in both of these genes suggests that these mutations might act as driver mutations of interest for further study. Disclosures: No relevant conflicts of interest to declare.

Supplementary file outlining the detailed sequencing methodology and additional figures. (DOCX 434 kb)

Genomic analysis of a rare human tumor Steven JM Jones, Janessa Laskin, Yvonne Y Li, Obi L Griffith, Jianghong An, Mikhail Bilenky, Yaron S Butterfield, Timothee Cezard, Eric Chuah, Richard Corbett, Anthony Fejes, Malachi Griffith, John Yee, Montgomery Martin, Michael Mayo, Nataliya Melnyk, Ryan D Morin, Trevor J Pugh, Tesa Severson, Sohrab P Shah, Margaret Sutcliffe, Angela Tam, Jefferson Terry, Nina Thiessen, Thomas Thomson, Richard Varhol, Thomas Zeng, Yongjun Zhao, Richard A Moore, David G Huntsman, Inanc Birol, Martin Hirst, Robert A Holt, Marco A Marra