Whole Genome and Exome Sequencing Reveals the Genetic Landscape of Burkitt Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 433-433
Author(s):  
Cassandra L Love ◽  
Dereje Jima ◽  
Jenny Zhang ◽  
Vladimir Grubor ◽  
Rodney R. Miles ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
High Throughput ◽  
Burkitt Lymphoma ◽  
Normal Tissue ◽  
High Throughput Sequencing ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Cancer Genes ◽  
Genetic Causes ◽  
Paired Normal Tissue

Abstract Abstract 433 Burkitt lymphoma (BL) is a relatively uncommon lymphoma, but is clinically important because it is curable when diagnosed properly. BL is also an important model disease for studying cancer. Chromosomal translocations of the MYC gene are a defining feature of BL. Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in adults and demonstrates overlapping morphology, immunophenotype and clinical behavior with BL. The genetic causes and the role of specific mutations in BL are largely unknown. Th e decoding of the human genome and the advent of high-throughput sequencing have provided rich opportunities for the comprehensive identification of the genetic causes of cancer. We began by sequencing 2 complete lymphoma tumor genomes (and paired normal tissue) derived from DLBCL and BL respectively. The pattern of somatic base alterations in both DLBCL and BL genomes indicated a predominance of G→A/C→T and A→G/T→C transitions (P<10−6) suggesting that the majority of these mutations arise from endogenous processes rather than environmental exposures, as has been observed with lung cancer and tobacco. In order to comprehensively identify genes that are recurrently mutated in DLBCL and BL, we obtained a total of 95 cases of DLBCLs and 60 cases of BL. The DLBCL cases were divided into a discovery set (N=34) and a prevalence set (N=61). The Burkitt cases were also divided into discovery and prevalence sets (N=15, N=45 respectively). For each of the discovery set cases we also obtained paired normal tissue. We performed whole-exome sequencing for all of these using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 6 GB of sequencing data using high throughput sequencing on the Illumina platform. We identified 525 candidate cancer genes that were recurrently somatically mutated in DLBCL and BL. We found that each tumor had an average of 20 gene alterations, which is fewer than most other solid tumors sequenced to date. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, PDGFRA) and chromatin modification (e.g. MLL3, SETD2), affecting 17.2% and 14.8% of the total genetic events respectively. We found several genes related to cancer that were commonly mutated in both BL and DLBCL, including MYC, BTG1 and SETD2. Mutations in MYC were much more common in BL compared to DLBCL, suggesting that mutation of MYC might serve as an independent oncogenic mechanism in BL, in addition to chromosomal translocations. Many known cancer genes were found to be exclusively mutated in BL including SMARCA4, a gene known to regulate the expression of CD44 which is implicated in tumorigenesis. This study represents one of the first in-depth analyses of a BL genome and one of the largest applications of exome sequencing in cancer. Our data provide the most comprehensive genetic portrait of human BL to date, and provides a significant first step to identifying the genetic causes of the disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals T cell neoepitope discovery in colorectal cancer by high throughput profiling of somatic mutations in expressed genes

Gut ◽  
2015 ◽  
Vol 66 (3) ◽  
pp. 454-463 ◽  
Author(s):  
Daniele Mennonna ◽  
Cristina Maccalli ◽  
Michele C Romano ◽  
Claudio Garavaglia ◽  
Filippo Capocefalo ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Epitope ◽  
Patient Specific ◽  
Cancer Genes ◽  
Healthy Donors

ObjectivePatient-specific (unique) tumour antigens, encoded by somatically mutated cancer genes, generate neoepitopes that are implicated in the induction of tumour-controlling T cell responses. Recent advancements in massive DNA sequencing combined with robust T cell epitope predictions have allowed their systematic identification in several malignancies.DesignWe undertook the identification of unique neoepitopes in colorectal cancers (CRCs) by using high-throughput sequencing of cDNAs expressed by standard cancer cell cultures, and by related cancer stem/initiating cells (CSCs) cultures, coupled with a reverse immunology approach not requiring human leukocyte antigen (HLA) allele-specific epitope predictions.ResultsSeveral unique mutated antigens of CRC, shared by standard cancer and related CSC cultures, were identified by this strategy. CD8+and CD4+T cells, either autologous to the patient or derived from HLA-matched healthy donors, were readily expanded in vitro by peptides spanning different cancer mutations and specifically recognised differentiated cancer cells and CSC cultures, expressing the mutations. Neoepitope-specific CD8+T cell frequency was also increased in a patient, compared with healthy donors, supporting the occurrence of clonal expansion in vivo.ConclusionsThese results provide a proof-of-concept approach for the identification of unique neoepitopes that are immunogenic in patients with CRC and can also target T cells against the most aggressive CSC component.

Exome Sequencing Reveals The Genetic Landscape Of Rare Atypical e6a2 CML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3995-3995
Author(s):  
Arati V. Rao ◽  
Yuri D. Fedoriw ◽  
Kristy L. Richards ◽  
Zhen Sun ◽  
Cassandra L Love ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Cancer Genes ◽  
Sequencing Data ◽  
Illumina Platform ◽  
Depth Analysis

Abstract Background Over 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML. Methodology We identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform. Results We identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs. Conclusion This study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival. Disclosures: No relevant conflicts of interest to declare.

High-Throughput Sequencing of the B Cell Receptor in African Burkitt Lymphoma Reveals Clues to Pathogenesis

immuneACCESS ◽  
2018 ◽  
Author(s):  
K Lombardo ◽  
D Coffey ◽  
A Morales ◽  
C Carlson ◽  
A Towlerton ◽  
...  
Keyword(s):  
B Cell ◽  
High Throughput ◽  
Burkitt Lymphoma ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
B Cell Receptor

scholarly journals Common Viral Integration Sites Identified in Avian Leukosis Virus-Induced B-Cell Lymphomas

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
James F. Justice ◽  
Robin W. Morgan ◽  
Karen L. Beemon
Keyword(s):  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Consensus Sequence ◽  
B Cell Lymphoma ◽  
Avian Leukosis Virus ◽  
B Cell Lymphomas ◽  
Genome Wide ◽  
Integration Sites

ABSTRACTAvian leukosis virus (ALV) induces B-cell lymphoma and other neoplasms in chickens by integrating within or near cancer genes and perturbing their expression. Four genes—MYC,MYB,Mir-155, andTERT—have previously been identified as common integration sites in these virus-induced lymphomas and are thought to play a causal role in tumorigenesis. In this study, we employ high-throughput sequencing to identify additional genes driving tumorigenesis in ALV-induced B-cell lymphomas. In addition to the four genes implicated previously, we identify other genes as common integration sites, includingTNFRSF1A,MEF2C,CTDSPL,TAB2,RUNX1,MLL5,CXorf57, andBACH2. We also analyze the genome-wide ALV integration landscapein vivoand find increased frequency of ALV integration near transcriptional start sites and within transcripts. Previous work has shown ALV prefers a weak consensus sequence for integration in cultured human cells. We confirm this consensus sequence for ALV integrationin vivoin the chicken genome.IMPORTANCEAvian leukosis virus induces B-cell lymphomas in chickens. Earlier studies showed that ALV can induce tumors through insertional mutagenesis, and several genes have been implicated in the development of these tumors. In this study, we use high-throughput sequencing to reveal the genome-wide ALV integration landscape in ALV-induced B-cell lymphomas. We find elevated levels of ALV integration near transcription start sites and use common integration site analysis to greatly expand the number of genes implicated in the development of these tumors. Interestingly, we identify several genes targeted by viral insertions that have not been previously shown to be involved in cancer.

scholarly journals Noninvasive monitoring of diffuse large B-cell lymphoma by immunoglobulin high-throughput sequencing

Blood ◽  
2015 ◽  
Vol 125 (24) ◽  
pp. 3679-3687 ◽  
Author(s):  
David M. Kurtz ◽  
Michael R. Green ◽  
Scott V. Bratman ◽  
Florian Scherer ◽  
Chih Long Liu ◽  
...  
Keyword(s):  
B Cell ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
High Specificity ◽  
Noninvasive Monitoring ◽  
Large B Cell Lymphoma ◽  
Key Points ◽  
Large B Cell

Key Points DLBCL can be detected in the blood by immunoglobulin high-throughput sequencing (Ig-HTS) with high specificity. Although DLBCL can be detected in leukocytes or plasma by Ig-HTS, plasma has greater sensitivity and more accurately reflects disease.

A Comprehensive Identification of the Microrna Transcriptome and Its Application in B Cell Malignancies.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2403-2403
Author(s):  
Cassandra L. Jacobs ◽  
Dereje D Jima ◽  
Jenny Zhang ◽  
Cherie Dunphy ◽  
Kristy L. Richards ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
High Throughput ◽  
Germinal Center ◽  
High Throughput Sequencing ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Microrna Expression ◽  
B Cell Malignancies

Abstract Abstract 2403 Poster Board II-380 Background MicroRNAs are 18-22 nucleotide-long RNA molecules that regulate expression of genes. We and others have previously demonstrated a role for microRNAs in the pathogenesis of B cell malignancies. Computational predictions suggest that the human genome encodes several thousand microRNAs. Thus far, about 700 microRNAs have been discovered in humans, including over 200 new microRNAs in the past year alone. The ongoing discovery of microRNAs makes it difficult to comprehensively study their role in a disease group. The advent of high throughput sequencing allows the simultaneous identification of millions of transcripts, thereby providing a sensitivity that is several orders of magnitude higher than conventional methods. We hypothesized that high throughput sequencing would be an effective tool to comprehensively identify microRNAs in normal and malignant B cells. While there is an overlap between diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) in morphology, immunophenotype and cytogenetics, distinguishing between BL and DLBCL is critical because there are important differences in their clinical management. We investigated whether microRNA expression could be used to reliably distinguish BL from DLBCL. Methods and Results We carefully chose 31 human samples to represent the spectrum of normal and malignant B cells including FACS-sorted naive, germinal center, memory, plasma cells, EBV transformed and activated B cells. Samples derived from B cell malignancies included B-lymphoblastic lymphoma, chronic lymphocytic leukemia (immunoglobulin gene mutated and unmutated), mantle cell lymphoma, marginal zone lymphomas, HIV-related lymphoma, BL, DLBCL (activated and germinal center type), primary mediastinal B cell lymphoma, Hodgkin lymphoma, and multiple myeloma. We applied massively parallel, high-throughput sequencing of the 18-22 nt RNAs from these cases and generated a total of 255,624,785 sequences (∼5 billion bases). Using a computational approach that we have previously validated with normal B cells, we identified the expression of 429 known microRNAs in normal and malignant B cells, a number that is over three times higher than previously recognized in any tissue type. We also identified the expression of 302 novel microRNAs in normal and malignant B cells. The vast majority of these microRNAs were highly conserved in multiple species. As a proof of principle, we generated a custom microarray that included all the known human, and viral microRNAs, as well as 302 novel microRNAs identified by sequencing, and applied it to the clinically important distinction of BL from DLBCL. Biopsy samples were collected from 104 patients (BL, N=25, DLBCL, N=79) treated at 9 institutions that comprise an international consortium. All cases were reviewed for pathology diagnosis and profiled for microRNA expression. We constructed a Bayesian predictor to distinguish BL from DLBCL based on the microRNA expression. The predictor performance was tested using leave-one-out cross-validation. We also applied gene expression profiling to the cases of DLBCL to identify the molecular subsets of DLBCL: activated B cell like and germinal center B cell like DLBCL. The microRNA profiles of these cases were equally efficacious in distinguishing the DLBCL subsets. The predictor constructed based on microRNA expression was over 90% accurate in distinguishing BL from DLBCL, using pathology diagnosis as the gold standard. Further, microRNA-based predictor was also over 90% accurate in the distinction of the molecular subsets of DLBCL, compared to the gold standard of gene expression-profiling. As additional validation, we performed in situ hybridization of selected microRNAs to directly visualize their expression using methods that are easily accessible in conventional pathology laboratories. We found excellent concordance between the expression results derived from microarrays and in situ hybridization suggesting a ready path to clinical translation. Conclusion Our study represents the first comprehensive delineation of microRNA expression in B cell malignancies using high throughput sequencing. Our data suggest that microRNAs are a promising marker for the distinction of aggressive lymphomas. Disclosures: No relevant conflicts of interest to declare.

Mechanisms in Leukemia-Associated Chromosomal Translocations: Novel, High-Throughput Sequencing Method to Detect Topoisomerase II (TOP2) Cleavage Complexes Genome-Wide with Exact Base Resolution,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3445-3445
Author(s):  
Carolyn A. Felix ◽  
Marie L. Carillo ◽  
Karen A. Urtishak ◽  
Isabelle C. Dragomir ◽  
Michele E. Paessler ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cord Blood ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Translocation Breakpoint ◽  
Chromosomal Translocations ◽  
Target Cells ◽  
Phosphodiester Bonds ◽  
Genome Wide ◽  
Translocation Breakpoints

Abstract Abstract 3445 Associations of chemotherapeutic TOP2 poisons with secondary leukemia have implicated TOP2-mediated DNA damage in balanced chromosomal translocations underlying many common forms of leukemia. “TOP2 poisons” convert native TOP2 into a cellular toxin by disrupting the cleavage/re-ligation equilibrium, either by decreasing the reverse rate of re-ligation or increasing the forward rate of cleavage, thus increasing cleavage complexes and causing DNA strand breaks that can promote recombination or initiate apoptosis. Besides anticancer chemotherapy, several dietary substances and the benzene metabolite p-benzoquinone are TOP2 poisons. Population and molecular epidemiology, translocation breakpoint junction sequences, temporal origins of translocations, and correlations of TOP2 in vitro cleavage sites with translocation breakpoints have pointed to a model in which TOP2 is the DNA damage mediator and resolution of TOP2 cleavage complexes, whether induced by chemotherapy, dietary substances, environmental toxins or ROS mediated damage, forms translocation breakpoint junctions. Still, the DNA damage mechanism(s) remain controversial. Investigation of cleavage complexes at the DNA sequence level in a human hematopoietic progenitor cell model that approximates target cells for translocations is the critical next step in testing this model. We invented a high-throughput sequencing-based method (Provisional Patent Filed) to address cause-and-effect relationships between TOP2 cleavage complexes and translocation breakpoints in the context of DNA topological structure in the chromatin of human hematopoietic cells. TOP2 relaxes supercoiled DNA by transiently cleaving and re-ligating both strands of the double helix. Each subunit of the TOP2 homodimer forms a phosphodiester bond with the base 3' to the cleavage. This creates a fleeting covalent TOP2-DNA intermediate called the cleavage complex with 4-base staggered DNA ends tethered by the enzyme. We implemented, refined, optimized and validated a novel assay system for immunodepletion of the alpha isoform of TOP2 (TOP2A) including DNA-bound TOP2A, with concomitant isolation and purification of the DNA from cleavage complexes in human hematopoietic cells. By taking advantage of the covalent phosphodiester bonds between TOP2A and DNA, the activity of calf intestinal phosphatase (CIP) (i.e. hydrolysis of phosphodiester bonds via removal of 5' phosphates) was used for a purpose never used before: to release DNA from cleavage complexes at exact sites of cleavage. These steps were performed in CEM cells and, to better mimic target cells for translocations, fresh cord blood mononuclear cells (MNCs) from three newborn infants. Western blot and Q-PCR analyses proved that we achieved: 1) isolation and immunodepletion of TOP2A and TOP2A-bound DNA, 2) CIP release of TOP2A-bound DNA from the cleavage complexes, and 3) quantitative enrichment of DNA amplicons near known MLL translocation breakpoint hotspots over that obtained using a negative control antibody for immunodepletion. By morphology and immunophenotype, the cord blood MNCs contained lymphocyte, monocyte, and minor CMP and GMP populations, and they mainly were in G0/G1 by cell cycle analysis. Even though TOP2A cleavage complex enrichment was evident, on Western blot analysis the TOP2A in the cord blood MNCs appeared predominantly degraded, which is consistent with known TOP2A cell cycle dependence and downregulation in quiescent cells. This validation forms the basis for the next steps in the assay to localize cleavage complexes at single base resolution genome-wide through high-throughput sequencing of DNA ends created by TOP2 and mapping them to the genome. This strategy comprises an entirely novel application of high-throughput sequencing with many possible future uses to define TOP2 cleavage complexes, as well as other adaptations to identify covalent DNA modifications with exact base precision. Secondary leukemias are a growing problem, and the incidence of infant leukemia where TOP2 poisons also have been implicated is increasing. Solving how DNA breaks arise and form chromosomal translocations would have profound implications for anticancer treatment and leukemia prevention. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Inference of Germline Mutational Status and Evaluation of Loss of Heterozygosity in High-Depth, Tumor-Only Sequencing Data

2018 ◽  
pp. 1-15 ◽  
Author(s):  
Hossein Khiabanian ◽  
Kim M. Hirshfield ◽  
Mendel Goldfinger ◽  
Simon Bird ◽  
Mark Stein ◽  
...  
Keyword(s):  
Loss Of Heterozygosity ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Familial Cancer ◽  
Cancer Genes ◽  
Sequencing Data ◽  
Tumor Purity ◽  
Statistical Model Selection ◽  
Mutational Status ◽  
High Depth

Purpose Inherited germline defects are implicated in up to 10% of human tumors, with particularly well-known roles in breast and ovarian cancers that harbor BRCA1/2-mutated genes. There is also increasing evidence for the role of germline alterations in other malignancies such as colon and pancreatic cancers. Mutations in familial cancer genes can be detected by high-throughput sequencing, when applied to formalin-fixed paraffin-embedded tumor specimens. However, because of the frequent lack of patient-matched control normal DNA and/or low tumor purity, there is limited ability to determine the genomic status of these alterations (germline v somatic) and to assess the presence of loss of heterozygosity (LOH). These analyses, especially when applied to genes such as BRCA1/2, can have significant clinical implications for patient care. Materials and Methods LOHGIC (LOH-germline inference calculator) is a statistical model selection method to determine somatic versus germline status and predict LOH for mutations identified via clinical grade, high-depth, hybrid capture, tumor-only sequencing. LOHGIC incorporates statistical uncertainties inherent to high-throughput sequencing as well as specimen biases in tumor purity estimates, which we used to assess BRCA1/2 mutations in 1,636 specimens sequenced at Rutgers Cancer Institute of New Jersey. Results Evaluation of LOHGIC with available germline sequencing from BRCA1/2 testing demonstrates 93% accuracy, 100% precision, and 96% recall. This analysis highlights a differential tumor spectrum associated with BRCA1/2 mutations. Conclusion LOHGIC can assess LOH status for both germline and somatic mutations. It also can be applied to any gene with candidate, inherited mutations. This approach demonstrates the clinical utility of targeted sequencing in both identifying patients with potential germline alterations in tumor suppressor genes as well as estimating LOH occurrence in cancer cells, which may confer therapeutic relevance.

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