scholarly journals Mutational landscape of canine B-cell lymphoma profiled at single nucleotide resolution by RNA-seq

PLoS ONE ◽  
2019 ◽  
Vol 14 (4) ◽  
pp. e0215154 ◽  
Author(s):  
Diana Giannuzzi ◽  
Laura Marconato ◽  
Luciano Cascione ◽  
Stefano Comazzi ◽  
Ramy Elgendy ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Rna Seq ◽  
Single Nucleotide ◽  
Mutational Landscape ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals Genome-wide DNA methylation and RNA-seq analyses identify genes and pathways associated with doxorubicin resistance in a canine diffuse large B-cell lymphoma cell line

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0250013
Author(s):  
Chia-Hsin Hsu ◽  
Hirotaka Tomiyasu ◽  
Chi-Hsun Liao ◽  
Chen-Si Lin
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Rna Seq ◽  
Doxorubicin Resistance ◽  
Large B Cell Lymphoma ◽  
Genome Wide ◽  
Large B Cell

Doxorubicin resistance is a major challenge in the successful treatment of canine diffuse large B-cell lymphoma (cDLBCL). In the present study, MethylCap-seq and RNA-seq were performed to characterize the genome-wide DNA methylation and differential gene expression patterns respectively in CLBL-1 8.0, a doxorubicin-resistant cDLBCL cell line, and in CLBL-1 as control, to investigate the underlying mechanisms of doxorubicin resistance in cDLBCL. A total of 20289 hypermethylated differentially methylated regions (DMRs) were detected. Among these, 1339 hypermethylated DMRs were in promoter regions, of which 24 genes showed an inverse correlation between methylation and gene expression. These 24 genes were involved in cell migration, according to gene ontology (GO) analysis. Also, 12855 hypermethylated DMRs were in gene-body regions. Among these, 353 genes showed a positive correlation between methylation and gene expression. Functional analysis of these 353 genes highlighted that TGF-β and lysosome-mediated signal pathways are significantly associated with the drug resistance of CLBL-1. The tumorigenic role of TGF-β signaling pathway in CLBL-1 8.0 was further validated by treating the cells with a TGF-β inhibitor(s) to show the increased chemo-sensitivity and intracellular doxorubicin accumulation, as well as decreased p-glycoprotein expression. In summary, the present study performed an integrative analysis of DNA methylation and gene expression in CLBL-1 8.0 and CLBL-1. The candidate genes and pathways identified in this study hold potential promise for overcoming doxorubicin resistance in cDLBCL.

scholarly journals Function annotation of the rice transcriptome at single-nucleotide resolution by RNA-seq

2010 ◽  
Vol 20 (9) ◽  
pp. 1238-1249 ◽  
Author(s):  
T. Lu ◽  
G. Lu ◽  
D. Fan ◽  
C. Zhu ◽  
W. Li ◽  
...  
Keyword(s):  
Rna Seq ◽  
Single Nucleotide ◽  
Function Annotation ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Mutations In Circadian Rhythm Genes Are Associated with Diffuse Large B-Cell Lymphoma In the Mexican Population

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2478-2478
Author(s):  
Miguel Ángel Gutiérrez-Monreal ◽  
Luis Villela ◽  
Yocanxochitl Perfecto ◽  
Samuel Kettenhofen ◽  
Guy Cardineau ◽  
...  
Keyword(s):  
Circadian Rhythm ◽  
High Risk ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Large B Cell Lymphoma ◽  
Risk Snps ◽  
Large B Cell

Abstract Abstract 2478 In Mexico, diffuse large B-cell lymphoma (DLBCL) is the most common histological non-Hodgkin's lymphoma (NHL) subtype and is observed in up to 48% of all diagnoses of lymphoma. It has recently been hypothesized that circadian disruption may have a connection with lymphomagenesis. The circadian clock maintains biological rhythms over a cycle of approximately 24 hours, and many physiological functions in the human body are regulated by this system. The disruption of the circadian cycle may negatively affect cellular function, potentially leading to increased susceptibility to certain malignancies. Evidence in support of this association has been provided by epidemiological studies that demonstrate that night-time work significantly increased the risk of NHL in men, the overall relative risk being 1.10 (95%CI = 1.03–1.19, p = 0.010)(Lahti et al., Int J Cancer 2008). The study was performed to determinate if functional single nucleotide polymorphisms (SNP) in circadian rhythm genes are associated with the presence of DLBCL in Mexican patients. A molecular mechanism to regulate the circadian rhythm has been identified, where nine genes have been involved: clock, npas2, bmal1, per1-3, cry1-2, and csnk1e. Recent studies have shown that approximately 10% of genes exhibit circadian patterns of expression in a given tissue, and 10% of these clock-controlled genes identified regulate either cell proliferation or apoptosis. For this current study, SNPs were evaluated in DNA samples isolated from histopathologically-confirmed DLBCL patients (N=30) and healthy volunteers (N=50) as a control population. Samples were provided by Hospital Regional de Especialidades IMSS No. 25 and Hospital Metropolitano, in Monterrey, Nuevo Leon, Mexico. The selection of functional SNPs was made using a bioinformatic tool called Function Analysis and Selection Tool for Single Nucleotide Polymorphisms (FASTSNP)(Yuan et al., Nucl. Acids Res 2006). Six genes that regulate the circadian rhythm were submitted to FASTSNP, and 13 high-risk SNPs were used for this study. The results provided were npas2 rs11541353, per1 rs3027189, rs3027180, and rs3027177; per2 rs2340885, and rs3739068; per3 rs228697, rs2640905, rs12078704, and rs57875989; tim rs2291739, and rs2638290; and cry2 rs2863712. Primers were designed for the 13 high-risk SNPs. The SNPs were amplified by PCR in a 96-well plate. Alleles were determined by presence of the resultant bands in an agarose gel electrophoresis. SNPs analysis showed that DLBCL patients were mutated in 93.3% (28/30), while the population of controls were only in 74.0% (37/50) who presented with mutations (p = 0.023). The rs2291739 variant of the tim gene was the most frequent homozygous mutant found in at least 50% of both controls and patients. Two SNPs were found to be statistically significant associated with DLBCL, these were per1 rs3027189 (p = 0.006) and cry2 rs2863712 (p = 0.002). The per1 gene plays an important role in regulating growth and DNA damage control and also interacts with proteins in the cell-cycle pathway. CRY2 has been shown to alter genes associated with immune response and hematological system development. These results suggest that these two genes might play an important role in influencing biological pathways relevant for lymphomagenesis. These findings contribute in part to the recent research that links the disruption of circadian rhythm with the processes of lymphomagenesis. Further studies are warranted. This study was supported by Catedra de Hematologia y Cancer from Tecnologico de Monterrey. Disclosures: No relevant conflicts of interest to declare.

Inactivating Gene Alterations of MHC Class II Transactivator CIITA Are Recurrent in Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 437-437
Author(s):  
Christian Steidl ◽  
Bruce Woolcock ◽  
Sanja Rogic ◽  
Susana Ben-Neriah ◽  
Adele Telenius ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Class Ii ◽  
Single Nucleotide ◽  
Coding Sequence ◽  
Breakpoint Cluster Region ◽  
Cluster Region ◽  
Gene Alterations

Abstract Abstract 437 INTRODUCTION: Escape from immune surveillance has been proposed to play an important role in cancer and in particular in the pathogenesis of a subgroup of lymphomas. Using massively parallel sequencing, we have recently identified recurrent rearrangements of the CIITA gene in classical Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL) (Steidl et al, Nature 2011). Specifically, a gene fusion in the HL cell line KM-H2, involving the genes CIITA and FLJ27352, suppressed expression of CIITA-regulated MHC class II genes in a dominant-negative manner. Therefore, we sought to further investigate if gene alterations targeting the CIITA locus are recurrent somatic events in HL and PMBCL that lead to loss of gene function and thereby reduce immunogenicity of the malignant cells. PATIENTS AND METHODS: To characterize CIITA gene alterations, we comprehensively studied the cell lines DEV (nodular lymphocyte predominance HL-derived) and KARPAS1106P (PMBCL-derived) by whole-transcriptome paired-end sequencing (RNA-seq) and single nucleotide polymorphism arrays (Affymetrix SNP 6.0). To identify single nucleotide mutations in the CIITA coding sequence we studied additional 7 mediastinal biopsy specimens of PMBCL by RNA-seq. Fluorescence in-situ hybridization (FISH) was used to determine and validate structural and copy number alterations of the CIITA locus. 23 samples of PMBCL, 3 PMBCL-derived cell lines and 9 HL cell lines were studied for mutations and small deletions in CIITA intron 1 by long-range PCR and direct sequencing. CIITA and HLA-DR expression levels were determined by quantitative reverse transcriptase PCR. RESULTS: We identified chromosomal rearrangements affecting both alleles of CIITA in DEV cells resulting in expression of CIITA-PDL2, CIITA-SOCS1 and SOCS1-CIITA fusion transcripts. In KARPAS1106P cells chromosome 16 deletions result in the loss of one entire CIITA allele and partial loss of the other allele. As a consequence, in both cell lines wildtype CIITA and HLA-DR expression was undetectable. Genomic breakpoints within the CIITA rearrangements, together with previously identified translocations, defined a 1.6Kb breakpoint cluster region in CIITA Intron 1. Further analysis of this breakpoint cluster region revealed small intronic deletions in 10 of 23 (43%) PMBCL cases while no such deletions were detected in 18 diffuse large B cell lymphoma and 15 reactive lymph node samples. Furthermore, sequencing revealed multiple deletions ranging from 1–1948 bp and a high incidence of single nucleotide mutations in the breakpoint cluster region of the deleted alleles. The base pair changes were enriched for C to T and G to A transitions over other transitions and transversions, indicative of a somatic hypermutation process. We found significantly lower CIITA expression in cases with CIITA rearrangement and/or small intronic deletions compared to cases without CIITA alterations (p=0.044). RNAseq revealed three cases with non-synonymous coding-sequence mutations including a truncating mutation in exon 4. These changes were somatic in 3 cases with available matching normal DNA. DISCUSSION:CIITA is the target of multiple genomic hits including chromosomal translocation, deletion and coding sequence mutations in HL and PMBCL. In two HL and PMBCL cell lines this leads to complete gene inactivation and loss of HLA class II expression. These data characterize CIITA alterations as a recurrent underlying genomic event of the previously described immunophenotype of reduced HLA class II expression in a subset of PMBCL cases. Our data show that an immune escape mechanism utilized by tumor cells can be directly linked to somatically acquired alterations in cancer genomes. Disclosures: Siebert: Abbott/Vysis: .

scholarly journals Mutational Landscape of Grey Zone Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 21-21
Author(s):  
Clementine Sarkozy ◽  
Stacy Hung ◽  
Katsuyoshi Takata ◽  
Elizabeth Chavez ◽  
Tomohiro Aoki ◽  
...  
Keyword(s):  
B Cell ◽  
Exome Sequencing ◽  
Board Of Directors ◽  
Research Funding ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Grey Zone ◽  
Advisory Committees ◽  
Mutational Landscape ◽  
Intermediate Morphology

Introduction: Grey zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classical Hodgkin lymphoma (cHL), is a rare and poorly defined entity. To decipher its mutational landscape and discover new therapeutic targets, we performed exome sequencing of 31 GZL cases. Methods: GZL cases from the LYSA group (N=139) and BC Cancer (N=30) were centrally reviewed and classified as previously published (Sarkozy et al, Am J Surg Pathol 2019). Whole-exome sequencing was performed on 31 cases with available fresh frozen tissue, using laser micro-dissection (LMD, MMI technology) to enrich for tumor cells and obtain matching normal DNA from microenvironment cells. DNA was extracted (Agencourt® DNAdvance kit) and genomic libraries were constructed with the Ovation ultra-low kit (Nugen®). Exome capture was performed using Agilent SureSelectXT V6+UTR followed by paired-end sequencing (NextSeq®). Somatic nucleotide variants (SNVs) and indels were identified using VarScan, Strelka and Mutect. Parameters affecting the sensitivity and specificity of variant calling were optimized using 7 "gold standard" cases for which DNA from peripheral blood cells was additionally available. Possible oncogenic drivers were identified based on rate of recurrence, MutSigCV and literature review. Results: Among the 31 GZL cases, the median age was 41 y (14-83) with a sex ratio of 15M:16F; 21 cases had mediastinal involvement, including 15 within the thymic area; EBER in-situ hybridization (ISH) was positive in 8 cases. Seven (23%) cases were classified as group-0 (cHL morphology with 100% CD20 expression), 22 (71%) with an intermediate morphology as group-1 (N=9, cHL-like morphology) or group-2 (N=13, LBCL-like morphology) and 2 (6%) as group-3 (LBCL with 100% of CD30 expression). The mean coverage was 96X (42-203) for tumor samples. One case was excluded due to failure in the LMD process. Among the 30 cases, 6628 variants across 4826 genes were found, including 2903 coding mutations (325 indels and 2808 SNVs, mean of 104/sample, range: 15-678), 721 affecting the 5' UTR and 2774 the 3' UTR. A total of 152 genes were identified as being potential oncogenic drivers, with a mean of 11 mutated genes per case (range 2-36). The most recurrently mutated genes were SOCS1 (33%), B2M (23%), GNA13 (20%), LRRN3 (17%), and ZNF217, NCOR1, ITPKB, IRF2BP2, CSF2RB, and CSMD3 (13% each). The epigenetic SWI/SNF and transcription regulation pathway (including NCOR1/2, ARID1A, KMT2D, KMT2A) was affected in 73% of the cases, JAK/STAT in 70% and NF-kB in 19%. As assessed by CNVkit and GISTIC, the most recurrent gains/amplifications identified were in 9p24.1 (JAK2, CD274, PDCD2LG2; 69%) and 2p16.1 (REL, BCL11A; 62%), and losses in 11q14.3 (ATM; 48%) and 12q24.33 (NCOR2; 48%). Based on mutational signature analysis, individual base substitutions were linked to mutagenic processes, with the highest contributions associated with aging (29%) and defective DNA mismatch repair (27%); moreover, mutations attributable to AID/APOBEC activity (5%), were found to be significantly enriched in EBV- vs. EBV+ cases (p = 0.013). EBV+ cases had fewer total variants (mean 98 vs 258, p=0.08) and potential oncogenic variants (mean 7 vs 15, p=0.03) compared to EBV- cases. EBV+ cases also lacked mutations in the NF-kB pathway and MHC-class I components (B2M and HLA-B: 0% vs 43% in EBV-, p=0.06) but had mutations in STAT3, DHX58, ACTB and ATP13A4 (6/7 cases) not present in the 23 EBV- cases. LRRN3 and GNA13 mutations were significantly associated with thymic area involvement (40% vs 0%, p=0.01). Furthermore, fluorescence-ISH indicated that 20% (1/5) of EBV+ cases had a rearrangement in the CIITA locus (16p13.13) vs 53% (9/17) in EBV- cases. Patients with an intermediate morphology had more oncogenic variants than those in group 0 and 3 (mean of 15 vs 6 variants/case, p=0.01 affecting 12 vs 5 genes, p=0.004). Finally, NCOR1 (N=4) and NCOR2 (N=2) mutations were exclusively found in cases with intermediate morphology (23% vs 0% for those with group 0 or 3 morphology). Conclusion: These data suggest that GZL is a highly heterogenous disease harboring somatic driver events shared with PMBCL and HL. We also discovered novel gene mutations pointing to the importance of previously unrecognized pathways in the pathogenesis of GZL. The distinct mutational pattern in EBV+ GZL suggests divergent evolutionary trajectories. Disclosures Sarkozy: Takeda: Research Funding. Salles:Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria; BMS: Honoraria; Amgen: Honoraria, Other: Educational events; Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events. Savage:BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Scott:Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Juno Therapeutics: Consultancy; Tioma: Research Funding; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Seattle Genetics: Consultancy; Bayer: Consultancy.

scholarly journals Antibody cross-reactivity accounts for widespread appearance of m1A in 5’ UTRs

2019 ◽  
Author(s):  
Anya V. Grozhik ◽  
Anthony O. Olarerin-George ◽  
Miriam Sindelar ◽  
Xing Li ◽  
Steven S. Gross ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Binding Sites ◽  
Cross Reactivity ◽  
Rna Seq ◽  
Transcription Start ◽  
Single Nucleotide ◽  
Biochemical Assays ◽  
Binding Antibody ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractN1-methyladenosine (m1A) was recently identified as a new mRNA modification based on its mapping to the 5’ UTRs of thousands of mRNAs with an m1A-binding antibody. More recent studies have confirmed the prevalence of m1A, while others have questioned it. To address this discrepancy, we mapped m1A using ultra-deep RNA-Seq datasets based on m1A-induced misincorporations during reverse transcription. Using this approach, we find m1A only in the mitochondrial MT-ND5 transcript. In contrast, when we mapped m1A antibody-binding sites at single-nucleotide resolution, we found binding to transcription start nucleotides in mRNA 5’ UTRs. Using different biochemical assays, we find that m1A is not present at these sites. Instead, we find that the m1A antibody exhibits m1A-independent binding to mRNA cap structures. We also tested a new and independently derived m1A antibody. We show that this m1A antibody lacks m7G cap-binding cross-reactivity, and notably does not map to 5’ UTRs in the transcriptome. Our data demonstrate that high-stoichiometry m1A sites are rare in the transcriptome and that previous mapping of m1A to mRNA 5’ UTRs are due to unintended binding of the m1A antibody to m7G cap structure in mRNA.

Mutational Landscape of Grey Zone Lymphoma

Blood ◽  
2020 ◽  
Author(s):  
Clementine Sarkozy ◽  
Stacy S. Hung ◽  
Elizabeth A. Chavez ◽  
Gerben Duns ◽  
Katsuyoshi Takata ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Anterior Mediastinal Mass ◽  
Grey Zone ◽  
Cell Of Origin ◽  
Disease Evolution ◽  
Mutational Landscape ◽  
Large B Cell Lymphoma ◽  
Large B Cell

The mutational landscape of grey zone lymphoma (GZL) has not yet been established and differences to related entities are largely unknown. Here, we studied coding sequence mutations of 50 EBV-negative GZL and 20 polymorphic EBV-positive DLBCL NOS (poly-EBV-L) in comparison to classical Hodgkin lymphoma (cHL), primary mediastinal large B cell lymphoma (PMBCL), and diffuse large B cell lymphoma (DLBCL). Exomes of 21 GZL and 7 poly-EBV-L cases along with paired normals were analyzed as a discovery cohort followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) displayed a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%) and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (N=18) had a significantly distinct pattern, enriched in mutations related to apoptosis defects (TP53 (39%), BCL2 (28%), BIRC6 (22%)) and depleted in GNA13, XPO1or NFKB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also presented more BCL2/BCL6 rearrangements as opposed to thymic GZL. Poly-EBV-L cases presented a distinct mutational profile including STAT3 mutations and a significantly lower coding-mutation load in comparison to EBV-negative GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche suggesting a common cell of origin with disease evolution overlapping with related anterior mediastinal lymphomas.

scholarly journals A refined cell-of-origin classifier with targeted NGS and artificial intelligence shows robust predictive value in DLBCL

2020 ◽  
Vol 4 (14) ◽  
pp. 3391-3404
Author(s):  
Zijun Y. Xu-Monette ◽  
Hongwei Zhang ◽  
Feng Zhu ◽  
Alexandar Tzankov ◽  
Govind Bhagat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Artificial Intelligence ◽  
Clinical Outcome ◽  
B Cell ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Survival Models ◽  
Rna Seq ◽  
Cell Of Origin ◽  
Training Set

Abstract Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell–like compared with the germinal-center B-cell–like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL.

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