Two Distinct Categories of Focal Deletions in Cancer Genomes.

One of the key questions about genomic alterations in cancer is whether they are functional in the sense of contributing to the selective advantage of tumor cells. The frequency with which an alteration occurs might reflect its ability to increase cancer cell growth, or alternatively, enhanced instability of a locus may increase the frequency with which it is found to be aberrant in tumors, regardless of oncogenic impact. Here we've addressed this on a genome-wide scale for cancer-associated focal deletions, which are known to pinpoint both tumor suppressor genes (tumor suppressors) and unstable loci. Based on DNA copy number analysis of over one-thousand human cancers representing ten different tumor types, we observed five loci with focal deletion frequencies above 5%, including the A2BP1 gene at 16p13.3 and the MACROD2 gene at 20p12.1. However, neither RNA expression nor functional studies support a tumor suppressor role for either gene. Further analyses suggest instead that these are sites of increased genomic instability and that they resemble common fragile sites (CFS). Genome-wide analysis revealed properties of CFS-like recurrent deletions that distinguish them from deletions affecting tumor suppressor genes, including their isolation at specific loci away from other genomic deletion sites, a considerably smaller deletion size, and dispersal throughout the affected locus rather than assembly at a common site of overlap. Additionally, CFS-like deletions have less impact on gene expression and are enriched in cell lines compared to primary tumors. We show that loci affected by CFS-like deletions are often distinct from known common fragile sites. Indeed, we find that each tumor tissue type has its own spectrum of CFS-like deletions, and that colon cancers have many more CFS-like deletions than other tumor types. We present simple rules that can pinpoint focal deletions that are not CFS-like and more likely to affect functional tumor suppressors.

Genetic heterogeneity of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma in adults. The disease exhibits a striking heterogeneity in gene expression profiles and clinical outcomes, but its genetic causes remain to be fully defined. Through whole genome and exome sequencing, we characterized the genetic diversity of DLBCL. In all, we sequenced 73 DLBCL primary tumors (34 with matched normal DNA). Separately, we sequenced the exomes of 21 DLBCL cell lines. We identified 322 DLBCL cancer genes that were recurrently mutated in primary DLBCLs. We identified recurrent mutations implicating a number of known and not previously identified genes and pathways in DLBCL including those related to chromatin modification (ARID1A and MEF2B), NF-κB (CARD11 and TNFAIP3), PI3 kinase (PIK3CD, PIK3R1, and MTOR), B-cell lineage (IRF8, POU2F2, and GNA13), and WNT signaling (WIF1). We also experimentally validated a mutation in PIK3CD, a gene not previously implicated in lymphomas. The patterns of mutation demonstrated a classic long tail distribution with substantial variation of mutated genes from patient to patient and also between published studies. Thus, our study reveals the tremendous genetic heterogeneity that underlies lymphomas and highlights the need for personalized medicine approaches to treating these patients.

The genetic landscape of mutations in Burkitt lymphoma.

Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Reducing system noise in copy number data using principal components of self-self hybridizations.

Genomic copy number variation underlies genetic disorders such as autism, schizophrenia, and congenital heart disease. Copy number variations are commonly detected by array based comparative genomic hybridization of sample to reference DNAs, but probe and operational variables combine to create correlated system noise that degrades detection of genetic events. To correct for this we have explored hybridizations in which no genetic signal is expected, namely "self-self" hybridizations (SSH) comparing DNAs from the same genome. We show that SSH trap a variety of correlated system noise present also in sample-reference (test) data. Through singular value decomposition of SSH, we are able to determine the principal components (PCs) of this noise. The PCs themselves offer deep insights into the sources of noise, and facilitate detection of artifacts. We present evidence that linear and piecewise linear correction of test data with the PCs does not introduce detectable spurious signal, yet improves signal-to-noise metrics, reduces false positives, and facilitates copy number determination.

Deep sequencing of the small RNA transcriptome of normal and malignant human B cells identifies hundreds of novel microRNAs.

A role for microRNA (miRNA) has been recognized in nearly every biologic system examined thus far. A complete delineation of their role must be preceded by the identification of all miRNAs present in any system. We elucidated the complete small RNA transcriptome of normal and malignant B cells through deep sequencing of 31 normal and malignant human B-cell samples that comprise the spectrum of B-cell differentiation and common malignant phenotypes. We identified the expression of 333 known miRNAs, which is more than twice the number previously recognized in any tissue type. We further identified the expression of 286 candidate novel miRNAs in normal and malignant B cells. These miRNAs were validated at a high rate (92%) using quantitative polymerase chain reaction, and we demonstrated their application in the distinction of clinically relevant subgroups of lymphoma. We further demonstrated that a novel miRNA cluster, previously annotated as a hypothetical gene LOC100130622, contains 6 novel miRNAs that regulate the transforming growth factor-ß pathway. Thus, our work suggests that more than a third of the miRNAs present in most cellular types are currently unknown and that these miRNAs may regulate important cellular functions.

Inferring tumor progression from genomic heterogeneity.

Cancer progression in humans is difficult to infer because we do not routinely sample patients at multiple stages of their disease. However, heterogeneous breast tumors provide a unique opportunity to study human tumor progression because they still contain evidence of early and intermediate subpopulations in the form of the phylogenetic relationships. We have developed a method we call Sector-Ploidy-Profiling (SPP) to study the clonal composition of breast tumors. SPP involves macro-dissecting tumors, flow-sorting genomic subpopulations by DNA content, and profiling genomes using comparative genomic hybridization (CGH). Breast carcinomas display two classes of genomic structural variation: (1) monogenomic and (2) polygenomic. Monogenomic tumors appear to contain a single major clonal subpopulation with a highly stable chromosome structure. Polygenomic tumors contain multiple clonal tumor subpopulations, which may occupy the same sectors, or separate anatomic locations. In polygenomic tumors, we show that heterogeneity can be ascribed to a few clonal subpopulations, rather than a series of gradual intermediates. By comparing multiple subpopulations from different anatomic locations, we have inferred pathways of cancer progression and the organization of tumor growth.

Novel genomic alterations and clonal evolution in chronic lymphocytic leukemia revealed by representational oligonucleotide microarray analysis (ROMA).

We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

Evaluation of the role of CYP6B cytochrome P450s in pyrethroid resistant Australian Helicoverpa armigera.

The AN02 strain of Helicoverpa armigera from eastern Australia exhibits 50-fold, PBO-suppressible resistance to the pyrethroid insecticide fenvalerate. The semidominant resistance gene RFen1 was previously mapped to AFLP Linkage Group 13. In evaluating the cytochrome P450 genes CYP6B7, CYP6B6, and CYP6B2 as candidates for RFen1, we found that they occur in a tandem array in the genome, next to the gene encoding the para-type sodium channel; the target of pyrethroid insecticides. We mapped these genes to AFLP Linkage Group 14, thus rejecting mutations within the P450 cluster or para as candidates for RFen1. RFen1 genotypes produced slightly different mRNA levels of the three P450s, but the differences were too small to convincingly account for resistance. We conclude that even if one or more of these P450s metabolize fenvalerate, they are unlikely to be responsible for the resistance in AN02.

Novel patterns of genome rearrangement and their association with survival in breast cancer.

Representational Oligonucleotide Microarray Analysis (ROMA) detects genomic amplifications and deletions with boundaries defined at a resolution of approximately 50 kb. We have used this technique to examine 243 breast tumors from two separate studies for which detailed clinical data were available. The very high resolution of this technology has enabled us to identify three characteristic patterns of genomic copy number variation in diploid tumors and to measure correlations with patient survival. One of these patterns is characterized by multiple closely spaced amplicons, or "firestorms," limited to single chromosome arms. These multiple amplifications are highly correlated with aggressive disease and poor survival even when the rest of the genome is relatively quiet. Analysis of a selected subset of clinical material suggests that a simple genomic calculation, based on the number and proximity of genomic alterations, correlates with life-table estimates of the probability of overall survival in patients with primary breast cancer. Based on this sample, we generate the working hypothesis that copy number profiling might provide information useful in making clinical decisions, especially regarding the use or not of systemic therapies (hormonal therapy, chemotherapy), in the management of operable primary breast cancer with ostensibly good prognosis, for example, small, node-negative, hormone-receptor-positive diploid cases.

PROBER: oligonucleotide FISH probe design software.

UNLABELLED: PROBER is an oligonucleotide primer design software application that designs multiple primer pairs for generating PCR probes useful for fluorescence in situ hybridization (FISH). PROBER generates Tiling Oligonucleotide Probes (TOPs) by masking repetitive genomic sequences and delineating essentially unique regions that can be amplified to yield small (100-2000 bp) DNA probes that in aggregate will generate a single, strong fluorescent signal for regions as small as a single gene. TOPs are an alternative to bacterial artificial chromosomes (BACs) that are commonly used for FISH but may be unstable, unavailable, chimeric, or non-specific to small (10-100 kb) genomic regions. PROBER can be applied to any genomic locus, with the limitation that the locus must contain at least 10 kb of essentially unique blocks. To test the software, we designed a number of probes for genomic amplifications and hemizygous deletions that were initially detected by Representational Oligonucleotide Microarray Analysis of breast cancer tumors. AVAILABILITY: http://prober.cshl.edu

Alternative splicing removes an Ets interaction domain from Lozenge during Drosophila eye development.

Physical and functional characteristics of the RUNX family of transcription factors are conserved between vertebrates and the Drosophila protein Lozenge. The runt-homology domain responsible for DNA binding and also the C-terminus are both nearly identical between the two proteins. The mammalian and fly proteins heterodimerize with a non-DNA binding partner protein to form a core binding factor essential for gene regulation during cell differentiation. The mammalian protein RUNX1 (AML1/PEBP2alphaB) interacts with the transcription factor Ets-1 to increase DNA binding and transactivation potential. Alternative splicing of the mammalian RUNX1 removes a domain required for this cooperative transactivation. In this work we determine the structure of the lozenge transcription unit and map 21 mutations. We show that the lozenge transcript is alternatively spliced during eye development to remove an Ets interaction domain. Emphasis is placed on Pointed the Drosophila homolog of the vertebrate Ets-1 protein; both Lozenge and Pointed proteins are needed for the activation of prospero expression. We use site-directed mutagenesis and yeast two-hybrid analysis to show that conserved amino acids within the alternate Lozenge exon are important for interaction with Pointed. Furthermore, the ectopic expression of Lozenge is sufficient to rescue Prospero expression in the presence of the Pointed competitor, Yan(ACT). We show that both lozenge isoforms are expressed during eye development and that the relative ratio of the transcripts for the two isoforms is sensitive to changes in Ras activity. We suggest that during eye development, Lozenge isoforms function in divergent roles, either interacting with Pointed on downstream targets or by functioning independently to establish distinct cell fates.