Publications by authors named "Barbara Meissner"

36 Publications

The impact of MYC and BCL2 structural variants in tumors of DLBCL morphology and mechanisms of false-negative MYC IHC.

Blood 2021 Apr;137(16):2196-2208

Centre for Lymphoid Cancer, BC Cancer, Vancouver, BC, Canada.

When the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.
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http://dx.doi.org/10.1182/blood.2020007193DOI Listing
April 2021

Coding and noncoding drivers of mantle cell lymphoma identified through exome and genome sequencing.

Blood 2020 07;136(5):572-584

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology.
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http://dx.doi.org/10.1182/blood.2019002385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440974PMC
July 2020

TMEM30A loss-of-function mutations drive lymphomagenesis and confer therapeutically exploitable vulnerability in B-cell lymphoma.

Nat Med 2020 04 24;26(4):577-588. Epub 2020 Feb 24.

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, British Columbia, Canada.

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.
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http://dx.doi.org/10.1038/s41591-020-0757-zDOI Listing
April 2020

The double-hit signature identifies double-hit diffuse large B-cell lymphoma with genetic events cryptic to FISH.

Blood 2019 10;134(18):1528-1532

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, Canada.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit-like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.
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http://dx.doi.org/10.1182/blood.2019002600DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6839951PMC
October 2019

Integrative genomic analysis identifies key pathogenic mechanisms in primary mediastinal large B-cell lymphoma.

Blood 2019 09 10;134(10):802-813. Epub 2019 Jul 10.

British Columbia Cancer, Centre for Lymphoid Cancer, Vancouver, BC, Canada.

Primary mediastinal large B-cell lymphoma (PMBL) represents a clinically and pathologically distinct subtype of large B-cell lymphomas. Furthermore, molecular studies, including global gene expression profiling, have provided evidence that PMBL is more closely related to classical Hodgkin lymphoma (cHL). Although targeted sequencing studies have revealed a number of mutations involved in PMBL pathogenesis, a comprehensive description of disease-associated genetic alterations and perturbed pathways is still lacking. Here, we performed whole-exome sequencing of 95 PMBL tumors to inform on oncogenic driver genes and recurrent copy number alterations. The integration of somatic gene mutations with gene expression signatures provides further insights into genotype-phenotype interrelation in PMBL. We identified highly recurrent oncogenic mutations in the Janus kinase-signal transducer and activator of transcription and nuclear factor κB pathways, and provide additional evidence of the importance of immune evasion in PMBL ( and ). Our analyses highlight the interferon response factor (IRF) pathway as a putative novel hallmark with frequent alterations in multiple pathway members ( and ). In addition, our integrative analysis illustrates the importance of and mutations driving oncogenic signaling. The identified driver genes were significantly more frequently mutated in PMBL compared with diffuse large B-cell lymphoma, whereas only a limited number of genes were significantly different between PMBL and cHL, emphasizing the close relation between these entities. Our study, performed on a large cohort of PMBL, highlights the importance of distinctive genetic alterations for disease taxonomy with relevance for diagnostic evaluation and therapeutic decision-making.
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http://dx.doi.org/10.1182/blood.2019001126DOI Listing
September 2019

Relapse of a group 4 medulloblastoma after 18 years as proven by histology and DNA methylation profiling.

Childs Nerv Syst 2019 06 22;35(6):1029-1033. Epub 2019 Feb 22.

Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246, Hamburg, Germany.

Background: Recent studies on medulloblastomas (MB) suggest that a large fraction of tumors appearing as late recurrence turn out to be secondary malignancies, e.g., malignant gliomas, after thorough molecular investigation.

Results: Here, we report of a patient with a group 4 MB that developed a distant recurrence after more than 18 years. The recurrent tumor was confirmed by histology and genome-wide DNA methylation profiling.

Conclusion: Our case not only illustrates the potential of very late recurrences after seemingly cured group 4 MB, but also illustrates that detailed molecular analyses are indispensable in patients with a history of a previous malignancy.
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http://dx.doi.org/10.1007/s00381-019-04086-3DOI Listing
June 2019

Molecular and Genetic Characterization of MHC Deficiency Identifies EZH2 as Therapeutic Target for Enhancing Immune Recognition.

Cancer Discov 2019 04 31;9(4):546-563. Epub 2019 Jan 31.

Centre for Lymphoid Cancer, British Columbia Cancer, Vancouver, British Columbia, Canada.

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II-deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of mutations in both MHC-I- and MHC-II-negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant . Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in -mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming. SIGNIFICANCE: We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin-specific context. Specifically, mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination...
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http://dx.doi.org/10.1158/2159-8290.CD-18-1090DOI Listing
April 2019

Double-Hit Gene Expression Signature Defines a Distinct Subgroup of Germinal Center B-Cell-Like Diffuse Large B-Cell Lymphoma.

J Clin Oncol 2019 01 3;37(3):190-201. Epub 2018 Dec 3.

1 British Columbia Cancer Centre for Lymphoid Cancer, Vancouver, British Columbia, Canada.

Purpose: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression.

Patients And Methods: We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data.

Results: We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested.

Conclusion: We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.
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http://dx.doi.org/10.1200/JCO.18.01583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6804880PMC
January 2019

High-resolution architecture and partner genes of rearrangements in lymphoma with DLBCL morphology.

Blood Adv 2018 10;2(20):2755-2765

Centre for Lymphoid Cancer, BC Cancer, Vancouver, BC, Canada.

Genomic rearrangements in the locus occur in ∼12% of lymphomas with diffuse large B-cell lymphoma (DLBCL) morphology and are associated with inferior outcome. Previous studies exploring rearrangements have primarily used fluorescence in situ hybridization (FISH) assays to characterize break-apart status but have rarely examined breakpoint location, and in some cases have not examined partner identity. We performed targeted sequencing of , , , and the immunoglobulin () loci in 112 tumors with DLBCL morphology harboring rearrangement. We characterized the location of the rearrangement at base pair resolution and identified the partner in 88 cases. We observed a cluster of breakpoints upstream of the coding region and in intron 1 (the "genic cluster"). Genic cluster rearrangements were enriched for translocations involving (80%), whereas nongenic rearrangements occurred mostly downstream of the gene with a variety of partners, including and Other recurrent partners included , , and , which has not previously been described as a partner. We compared 2 commercially available FISH break-apart assays for the locus and observed discordant results in 32% of cases examined, including some with - and - rearrangements. In cases of high-grade B-cell lymphoma with and and/or rearrangement (HGBL-DH), so-called "double-hit" lymphomas, the majority of rearrangements had non- partners (65%), with breakpoints outside the genic cluster (72%). In patients with de novo HGBL-DH of DLBCL morphology, - rearrangements showed a trend toward inferior time to progression and overall survival compared with -non- rearrangements. Our data reveal clinically relevant architecture of rearrangements in lymphomas with DLBCL morphology.
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http://dx.doi.org/10.1182/bloodadvances.2018023572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199666PMC
October 2018

Genome-wide discovery of somatic regulatory variants in diffuse large B-cell lymphoma.

Nat Commun 2018 10 1;9(1):4001. Epub 2018 Oct 1.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.
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http://dx.doi.org/10.1038/s41467-018-06354-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6167379PMC
October 2018

Assessment of Capture and Amplicon-Based Approaches for the Development of a Targeted Next-Generation Sequencing Pipeline to Personalize Lymphoma Management.

J Mol Diagn 2018 03 8;20(2):203-214. Epub 2018 Feb 8.

Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, British Columbia, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada. Electronic address:

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management.
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http://dx.doi.org/10.1016/j.jmoldx.2017.11.010DOI Listing
March 2018

Genetic profiling of and in diffuse large B-cell lymphoma determines cell-of-origin-specific clinical impact.

Blood 2017 05 28;129(20):2760-2770. Epub 2017 Mar 28.

Centre for Lymphoid Cancer.

The clinical significance of and genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled and genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of / genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of gain/amplification is significantly associated with poor outcome in activated B-cell-like and translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.
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http://dx.doi.org/10.1182/blood-2016-11-747022DOI Listing
May 2017

Histological Transformation and Progression in Follicular Lymphoma: A Clonal Evolution Study.

PLoS Med 2016 Dec 13;13(12):e1002197. Epub 2016 Dec 13.

Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada.

Background: Follicular lymphoma (FL) is an indolent, yet incurable B cell malignancy. A subset of patients experience an increased mortality rate driven by two distinct clinical end points: histological transformation and early progression after immunochemotherapy. The nature of tumor clonal dynamics leading to these clinical end points is poorly understood, and previously determined genetic alterations do not explain the majority of transformed cases or accurately predict early progressive disease. We contend that detailed knowledge of the expansion patterns of specific cell populations plus their associated mutations would provide insight into therapeutic strategies and disease biology over the time course of FL clinical histories.

Methods And Findings: Using a combination of whole genome sequencing, targeted deep sequencing, and digital droplet PCR on matched diagnostic and relapse specimens, we deciphered the constituent clonal populations in 15 transformation cases and 6 progression cases, and measured the change in clonal population abundance over time. We observed widely divergent patterns of clonal dynamics in transformed cases relative to progressed cases. Transformation specimens were generally composed of clones that were rare or absent in diagnostic specimens, consistent with dramatic clonal expansions that came to dominate the transformation specimens. This pattern was independent of time to transformation and treatment modality. By contrast, early progression specimens were composed of clones that were already present in the diagnostic specimens and exhibited only moderate clonal dynamics, even in the presence of immunochemotherapy. Analysis of somatic mutations impacting 94 genes was undertaken in an extension cohort consisting of 395 samples from 277 patients in order to decipher disrupted biology in the two clinical end points. We found 12 genes that were more commonly mutated in transformed samples than in the preceding FL tumors, including TP53, B2M, CCND3, GNA13, S1PR2, and P2RY8. Moreover, ten genes were more commonly mutated in diagnostic specimens of patients with early progression, including TP53, BTG1, MKI67, and XBP1.

Conclusions: Our results illuminate contrasting modes of evolution shaping the clinical histories of transformation and progression. They have implications for interpretation of evolutionary dynamics in the context of treatment-induced selective pressures, and indicate that transformation and progression will require different clinical management strategies.
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http://dx.doi.org/10.1371/journal.pmed.1002197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5154502PMC
December 2016

Cell of origin of transformed follicular lymphoma.

Blood 2015 Oct 25;126(18):2118-27. Epub 2015 Aug 25.

Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, and.

Follicular lymphoma (FL) is an indolent disease but transforms in 2% to 3% of patients per year into aggressive, large cell lymphoma, a critical event in the course of the disease associated with increased lymphoma-related mortality. Early transformation cannot be accurately predicted at the time of FL diagnosis and the biology of transformed FL (TFL) is poorly understood. Here, we assembled a cohort of 126 diagnostic FL specimens including 40 patients experiencing transformation (<5 years) and 86 patients not experiencing transformation for at least 5 years. In addition, we assembled an overlapping cohort of 155 TFL patients, including 114 cases for which paired samples were available, and assessed temporal changes of routinely available biomarkers, outcome after transformation, as well as molecular subtypes of TFL. We report that the expression of IRF4 is an independent predictor of early transformation (Hazard ratio, 13.3; P < .001). We also show that composite histology at the time of transformation predicts favorable prognosis. Moreover, applying the Lymph2Cx digital gene expression assay for diffuse large B-cell lymphoma (DLBCL) cell-of-origin determination to 110 patients with DLBCL-like TFL, we demonstrate that TFL is of the germinal-center B-cell-like subtype in the majority of cases (80%) but that a significant proportion of cases is of the activated B-cell-like (ABC) subtype (16%). These latter cases are commonly negative for BCL2 translocation and arise preferentially from BCL2 translocation-negative and/or IRF4-expressing FLs. Our study demonstrates the existence of molecular heterogeneity in TFL as well as its relationship to the antecedent FL.
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http://dx.doi.org/10.1182/blood-2015-06-649905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626253PMC
October 2015

Prognostic Significance of Diffuse Large B-Cell Lymphoma Cell of Origin Determined by Digital Gene Expression in Formalin-Fixed Paraffin-Embedded Tissue Biopsies.

J Clin Oncol 2015 Sep 3;33(26):2848-56. Epub 2015 Aug 3.

David W. Scott, Anja Mottok, Daisuke Ennishi, Pedro Farinha, Susana Ben-Neriah, Robert Kridel, Garrett S. Barry, Christoffer Hother, Pau Abrisqueta, Merrill Boyle, Barbara Meissner, Adele Telenius, Kerry J. Savage, Laurie H. Sehn, Graham W. Slack, Christian Steidl, Joseph M. Connors, and Randy D. Gascoyne, British Columbia Cancer Agency, Vancouver, British Columbia, Canada; George W. Wright and Louis M. Staudt, National Cancer Institute, Bethesda, MD; and Lisa M. Rimsza, University of Arizona, Tucson, AZ.

Purpose: To evaluate the prognostic impact of cell-of-origin (COO) subgroups, assigned using the recently described gene expression-based Lymph2Cx assay in comparison with International Prognostic Index (IPI) score and MYC/BCL2 coexpression status (dual expressers).

Patients And Methods: Reproducibility of COO assignment using the Lymph2Cx assay was tested employing repeated sampling within tumor biopsies and changes in reagent lots. The assay was then applied to pretreatment formalin-fixed paraffin-embedded tissue (FFPET) biopsies from 344 patients with de novo diffuse large B-cell lymphoma (DLBCL) uniformly treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) at the British Columbia Cancer Agency. MYC and BCL2 protein expression was assessed using immunohistochemistry on tissue microarrays.

Results: The Lymph2Cx assay provided concordant COO calls in 96% of 49 repeatedly sampled tumor biopsies and in 100% of 83 FFPET biopsies tested across reagent lots. Critically, no frank misclassification (activated B-cell-like DLBCL to germinal center B-cell-like DLBCL or vice versa) was observed. Patients with activated B-cell-like DLBCL had significantly inferior outcomes compared with patients with germinal center B-cell-like DLBCL (log-rank P < .001 for time to progression, progression-free survival, disease-specific survival, and overall survival). In pairwise multivariable analyses, COO was associated with outcomes independent of IPI score and MYC/BCL2 immunohistochemistry. The prognostic significance of COO was particularly evident in patients with intermediate IPI scores and the non-MYC-positive/BCL2-positive subgroup (log-rank P < .001 for time to progression).

Conclusion: Assignment of DLBCL COO by the Lymph2Cx assay using FFPET biopsies identifies patient groups with significantly different outcomes after R-CHOP, independent of IPI score and MYC/BCL2 dual expression.
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http://dx.doi.org/10.1200/JCO.2014.60.2383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4554747PMC
September 2015

Pharmacological and genomic profiling identifies NF-κB-targeted treatment strategies for mantle cell lymphoma.

Nat Med 2014 Jan 22;20(1):87-92. Epub 2013 Dec 22.

1] Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA. [2].

Mantle cell lymphoma (MCL) is an aggressive malignancy that is characterized by poor prognosis. Large-scale pharmacological profiling across more than 100 hematological cell line models identified a subset of MCL cell lines that are highly sensitive to the B cell receptor (BCR) signaling inhibitors ibrutinib and sotrastaurin. Sensitive MCL models exhibited chronic activation of the BCR-driven classical nuclear factor-κB (NF-κB) pathway, whereas insensitive cell lines displayed activation of the alternative NF-κB pathway. Transcriptome sequencing revealed genetic lesions in alternative NF-κB pathway signaling components in ibrutinib-insensitive cell lines, and sequencing of 165 samples from patients with MCL identified recurrent mutations in TRAF2 or BIRC3 in 15% of these individuals. Although they are associated with insensitivity to ibrutinib, lesions in the alternative NF-κB pathway conferred dependence on the protein kinase NIK (also called mitogen-activated protein 3 kinase 14 or MAP3K14) both in vitro and in vivo. Thus, NIK is a new therapeutic target for MCL treatment, particularly for lymphomas that are refractory to BCR pathway inhibitors. Our findings reveal a pattern of mutually exclusive activation of the BCR-NF-κB or NIK-NF-κB pathways in MCL and provide critical insights into patient stratification strategies for NF-κB pathway-targeted agents.
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http://dx.doi.org/10.1038/nm.3435DOI Listing
January 2014

Frequent and sex-biased deletion of SLX4IP by illegitimate V(D)J-mediated recombination in childhood acute lymphoblastic leukemia.

Hum Mol Genet 2014 Feb 17;23(3):590-601. Epub 2013 Sep 17.

Department of Pediatrics, University Hospital Schleswig-Holstein, Kiel, Germany.

Acute lymphoblastic leukemia (ALL) accounts for ∼25% of pediatric malignancies. Of interest, the incidence of ALL is observed ∼20% higher in males relative to females. The mechanism behind the phenomenon of sex-specific differences is presently not understood. Employing genome-wide genetic aberration screening in 19 ALL samples, one of the most recurrent lesions identified was monoallelic deletion of the 5' region of SLX4IP. We characterized this deletion by conventional molecular genetic techniques and analyzed its interrelationships with biological and clinical characteristics using specimens and data from 993 pediatric patients enrolled into trial AIEOP-BFM ALL 2000. Deletion of SLX4IP was detected in ∼30% of patients. Breakpoints within SLX4IP were defined to recurrent positions and revealed junctions with typical characteristics of illegitimate V(D)J-mediated recombination. In initial and validation analyses, SLX4IP deletions were significantly associated with male gender and ETV6/RUNX1-rearranged ALL (both overall P < 0.0001). For mechanistic validation, a second recurrent deletion affecting TAL1 and caused by the same molecular mechanism was analyzed in 1149 T-cell ALL patients. Validating a differential role by sex of illegitimate V(D)J-mediated recombination at the TAL1 locus, 128 out of 1149 T-cell ALL samples bore a deletion and males were significantly more often affected (P = 0.002). The repeatedly detected association of SLX4IP deletion with male sex and the extension of the sex bias to deletion of the TAL1 locus suggest that differential illegitimate V(D)J-mediated recombination events at specific loci may contribute to the consistent observation of higher incidence rates of childhood ALL in boys compared with girls.
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http://dx.doi.org/10.1093/hmg/ddt447DOI Listing
February 2014

Mutational and structural analysis of diffuse large B-cell lymphoma using whole-genome sequencing.

Blood 2013 Aug 22;122(7):1256-65. Epub 2013 May 22.

Genome Sciences Centre, BC Cancer Agency, Vancouver, Canada.

Diffuse large B-cell lymphoma (DLBCL) is a genetically heterogeneous cancer composed of at least 2 molecular subtypes that differ in gene expression and distribution of mutations. Recently, application of genome/exome sequencing and RNA-seq to DLBCL has revealed numerous genes that are recurrent targets of somatic point mutation in this disease. Here we provide a whole-genome-sequencing-based perspective of DLBCL mutational complexity by characterizing 40 de novo DLBCL cases and 13 DLBCL cell lines and combining these data with DNA copy number analysis and RNA-seq from an extended cohort of 96 cases. Our analysis identified widespread genomic rearrangements including evidence for chromothripsis as well as the presence of known and novel fusion transcripts. We uncovered new gene targets of recurrent somatic point mutations and genes that are targeted by focal somatic deletions in this disease. We highlight the recurrence of germinal center B-cell-restricted mutations affecting genes that encode the S1P receptor and 2 small GTPases (GNA13 and GNAI2) that together converge on regulation of B-cell homing. We further analyzed our data to approximate the relative temporal order in which some recurrent mutations were acquired and demonstrate that ongoing acquisition of mutations and intratumoral clonal heterogeneity are common features of DLBCL. This study further improves our understanding of the processes and pathways involved in lymphomagenesis, and some of the pathways mutated here may indicate new avenues for therapeutic intervention.
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http://dx.doi.org/10.1182/blood-2013-02-483727DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3744992PMC
August 2013

Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons.

Biochim Biophys Acta 2013 Jun 27;1833(6):1421-33. Epub 2013 Feb 27.

Division of Neurobiochemistry, Biocenter, Innsbruck Medical University, Innsbruck, Austria.

RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [(3)H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca(2+) oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca(2+) dynamics the in neurons.
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http://dx.doi.org/10.1016/j.bbamcr.2013.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636420PMC
June 2013

The E3 ubiquitin ligase UBR5 is recurrently mutated in mantle cell lymphoma.

Blood 2013 Apr 13;121(16):3161-4. Epub 2013 Feb 13.

Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, BC, Canada V5Z 1L3.

We have recently reported the application of RNAseq to mantle cell lymphoma (MCL) transcriptomes revealing recurrent mutations in NOTCH1. Here we describe the targeted resequencing of 18 genes mutated in this discovery cohort using a larger cohort of MCL tumors. In addition to frequent mutations in ATM, CCND1, TP53, and NOTCH1, mutations were also observed recurrently in MEF2B, TRAF2, and TET2. Interestingly, the third most frequently mutated gene was UBR5, a gene encoding a 2799aa protein, with multiple functions, including E3 ligase activity based on a conserved cysteine residue at the C-terminus. Nonsynonymous mutations were detected in 18% (18/102) of tumors, with 61% of the mutations resulting in frameshifts in, or around, exon 58, predicted to result in the loss of this conserved cysteine residue. The recurrence and clustering of deleterious mutations implicate UBR5 mutations as a critical pathogenic event in a subgroup of MCL.
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http://dx.doi.org/10.1182/blood-2013-01-478834DOI Listing
April 2013

Gene expression-based model using formalin-fixed paraffin-embedded biopsies predicts overall survival in advanced-stage classical Hodgkin lymphoma.

J Clin Oncol 2013 Feb 26;31(6):692-700. Epub 2012 Nov 26.

Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, British Columbia, Canada.

Purpose: Our aim was to reliably identify patients with advanced-stage classical Hodgkin lymphoma (cHL) at increased risk of death by developing a robust predictor of overall survival (OS) using gene expression measured in routinely available formalin-fixed paraffin-embedded tissue (FFPET).

Methods: Expression levels of 259 genes, including those previously reported to be associated with outcome in cHL, were determined by digital expression profiling of pretreatment FFPET biopsies from 290 patients enrolled onto the E2496 Intergroup trial comparing doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) and Stanford V regimens in locally extensive and advanced-stage cHL. A model for OS separating patients into low- and high-risk groups was produced using penalized Cox regression. The model was tested in an independent cohort of 78 patients enriched for treatment failure but otherwise similar to patients in a population-based registry of patients treated with ABVD. Weighted analysis methods generated unbiased estimates of predictor performance in the population-based registry.

Results: A 23-gene outcome predictor was generated. The model identified a population at increased risk of death in the validation cohort. There was a 29% absolute difference in 5-year OS between the high- and low-risk groups (63% v 92%, respectively; log-rank P < .001; hazard ratio, 6.7; 95% CI, 2.6 to 17.4). The predictor was superior to the International Prognostic Score and CD68 immunohistochemistry in multivariate analyses.

Conclusion: A gene expression-based predictor, developed in and applicable to routinely available FFPET biopsies, identifies patients with advanced-stage cHL at increased risk of death when treated with standard-intensity up-front regimens.
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http://dx.doi.org/10.1200/JCO.2012.43.4589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3574267PMC
February 2013

Ikaros (IKZF1) alterations and minimal residual disease at day 15 assessed by flow cytometry predict prognosis of childhood BCR/ABL-negative acute lymphoblastic leukemia.

Pediatr Blood Cancer 2013 Mar 19;60(3):420-7. Epub 2012 Sep 19.

2nd Faculty of Medicine, Department of Pediatric Hematology and Oncology, Charles University and University Hospital Motol, Prague, Czech Republic.

Background: Recently, several studies have demonstrated a negative prognostic impact of Ikaros (IKZF1) gene alterations in acute lymphoblastic leukemia (ALL). However, controversies still exist regarding the impact of IKZF1 in current treatment protocols.

Procedure: We simultaneously detected IKZF1 gene deletions by multiplex ligation-dependent probe amplification and gene expression of IKZF1 isoforms in 206 children with BCR/ABL-negative ALL treated with ALL IC-BFM 2002 protocol, in which risk stratification was not based on minimal residual disease (MRD), and validated the results on a cohort of 189 patients treated with MRD-directed ALL-BFM 2000 protocol.

Results: Deletion of IKZF1 was present in 14 of 206 (7%) ALL IC patients. Interestingly, gene expression did not completely correlate with the deletion status in either cohort. Deletions were not always reflected in the gene expression of dominant-negative isoforms, and conversely, 7 of 395 (2%) non-deleted cases overexpressed dominant-negative isoform Ik6. IKZF1 deletions significantly affected event-free survival (EFS) of the ALL IC cohort (41 ± 14% vs. 86 ± 3%, P < 0.0001). Regarding IKZF1 isoforms, only Ik6 overexpression had negative prognostic impact (EFS 50 ± 16% vs. 85 ± 3%, P = 0.003). In multivariate analysis, which included ALL IC risk criteria, flow-cytometric MRD and IKZF1 alterations, day 15 MRD and IKZF1 deletion status displayed an independent prognostic impact.

Conclusions: We show that MRD-directed treatment diminishes prognostic impact of IKZF1 alterations. However, IKZF1 status alone or combined with day 15 flow cytometry can significantly improve risk stratification within BFM protocols at centers that do not perform antigen-receptor-based MRD monitoring.
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http://dx.doi.org/10.1002/pbc.24299DOI Listing
March 2013

IKZF1 deletion is an independent predictor of outcome in pediatric acute lymphoblastic leukemia treated according to the ALL-BFM 2000 protocol.

Haematologica 2013 Mar 8;98(3):428-32. Epub 2012 Aug 8.

Department of Pediatrics, University Medical Centre Schleswig-Holstein, Kiel, Germany.

IKZF1 gene deletions have been associated with a poor outcome in pediatric precursor B-cell acute lymphoblastic leukemia. To assess the prognostic relevance of IKZF1 deletions for patients treated on Berlin-Frankfurt-Münster Study Group trial ALL-BFM 2000, we screened 694 diagnostic acute lymphoblastic leukemia samples by Multiplex Ligation-dependent Probe Amplification. Patients whose leukemic cells bore IKZF1 deletions had a lower 5-year event-free survival (0.69±0.05 vs. 0.85±0.01; P<0.0001) compared to those without, mainly due to a higher cumulative incidence of relapses (0.21±0.04 vs. 0.10±0.01; P=0.001). Although IKZF1 deletions were significantly associated with the P2RY8-CRLF2 rearrangement, their prognostic value was found to be independent from this association. Thus, IKZF1 deletion is an independent predictor of treatment outcome and a strong candidate marker for integration in future treatment stratification strategies on ALL-BFM protocols. Clinicaltrials.gov identifier: NCT00430118.
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http://dx.doi.org/10.3324/haematol.2011.056135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3659952PMC
March 2013

Prediction of outcome by early bone marrow response in childhood acute lymphoblastic leukemia treated in the ALL-BFM 95 trial: differential effects in precursor B-cell and T-cell leukemia.

Haematologica 2012 Jul 22;97(7):1048-56. Epub 2012 Jan 22.

Pediatric Hematology and Oncology, University Hospital Schleswig-Holstein, Lübeck Campus, Germany.

Background: In the ALL-BFM 95 trial for treatment of acute lymphoblastic leukemia, response to a prednisone pre-phase (prednisone response) was used for risk stratification in combination with age and white blood cell count at diagnosis, response to induction therapy and specific genetic high-risk features.

Design And Methods: Cytomorphological marrow response was prospectively assessed on Day 15 during induction, and its prognostic value was analyzed in 1,431 patients treated on ALL-BFM 95.

Results: The 8-year probabilities of event-free survival were 86.1%, 74.5%, and 46.4% for patients with M1, M2, and M3 Day 15 marrows, respectively. Compared to prednisone response, Day 15 marrow response was superior in outcome prediction in precursor B-cell and T-cell leukemia with, however, a differential effect depending on blast lineage. Outcome was poor in T-cell leukemia patients with prednisone poor-response independent of Day 15 marrow response, whereas among patients with prednisone good-response different risk groups could be identified by Day 15 marrow response. In contrast, prednisone response lost prognostic significance in precursor B-cell leukemia when stratified by Day 15 marrow response. Age and white blood cell count retained their independent prognostic effect.

Conclusions: Selective addition of Day 15 marrow response to conventional stratification criteria applied on ALL-BFM 95 (currently in use in several countries as regular chemotherapy protocol for childhood acute lymphoblastic leukemia) may significantly improve risk-adapted treatment delivery. Even though cutting-edge trial risk stratification is meanwhile dominated by minimal residual disease evaluation, an improved conventional risk assessment, as presented here, could be of great importance to countries that lack the technical and/or financial resources associated with the application of minimal residual disease analysis.
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http://dx.doi.org/10.3324/haematol.2011.047613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396677PMC
July 2012

Whole transcriptome sequencing reveals recurrent NOTCH1 mutations in mantle cell lymphoma.

Blood 2012 Mar 30;119(9):1963-71. Epub 2011 Dec 30.

Centre for Lymphoid Cancer, British Columbia Cancer Agency, 675 W.10th Ave.,Vancouver, BC, Canada.

Mantle cell lymphoma (MCL), an aggressive subtype of non-Hodgkin lymphoma, is characterized by the hallmark translocation t(11;14)(q13;q32) and the resulting overexpression of cyclin D1 (CCND1). Our current knowledge of this disease encompasses frequent secondary cytogenetic aberrations and the recurrent mutation of a handful of genes, such as TP53, ATM, and CCND1. However, these findings insufficiently explain the biologic underpinnings of MCL. Here, we performed whole transcriptome sequencing on a discovery cohort of 18 primary tissue MCL samples and 2 cell lines. We found recurrent mutations in NOTCH1, a finding that we confirmed in an extension cohort of 108 clinical samples and 8 cell lines. In total, 12% of clinical samples and 20% of cell lines harbored somatic NOTCH1 coding sequence mutations that clustered in the PEST domain and predominantly consisted of truncating mutations or small frame-shifting indels. NOTCH1 mutations were associated with poor overall survival (P = .003). Furthermore, we showed that inhibition of the NOTCH pathway reduced proliferation and induced apoptosis in 2 MCL cell lines. In summary, we have identified recurrent NOTCH1 mutations that provide the preclinical rationale for therapeutic inhibition of the NOTCH pathway in a subset of patients with MCL.
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http://dx.doi.org/10.1182/blood-2011-11-391474DOI Listing
March 2012

Low platelet counts after induction therapy for childhood acute lymphoblastic leukemia are strongly associated with poor early response to treatment as measured by minimal residual disease and are prognostic for treatment outcome.

Haematologica 2012 Mar 4;97(3):402-9. Epub 2011 Nov 4.

Department of Molecular Hematopoiesis, Hannover Medical School, Hannover, Germany.

Background: Numerous reports have been published on the association between kinetics of leukemic cells during early treatment of childhood acute lymphoblastic leukemia and therapeutic outcome. In contrast, little is known about the prognostic relevance of normal blood counts in this setting.

Design And Methods: Normal hematopoiesis during and after induction treatment (days 8, 15 and 33) was correlated with therapeutic outcome in a cohort of 256 children with acute lymphoblastic leukemia treated in one of three consecutive ALL-BFM trials at a single institute. Replication analysis of positive findings was performed in an independent cohort of 475 patients from the ALL-BFM 2000 multicenter trial.

Results: A platelet count in the first quartile on treatment day 33 and a neutrophil count above the median on day 8 were significantly associated with treatment outcome, conferring multivariate risk ratios for an event of 3.27 (95% confidence interval 1.60-6.69) and 2.26 (95% confidence interval 1.23-4.29), respectively. Replication analysis confirmed the prognostic effect of platelet count on treatment day 33 and demonstrated a strong association with minimal residual disease-based risk group distribution (P<0.00001).

Conclusions: Platelet counts after induction treatment may improve treatment stratification for patients with childhood acute lymphoblastic leukemia and be of particular interest in non-minimal residual disease-based trials. (ALL-BFM 2000 is registered at: ClinicalTrials.gov: NCT00430118. National Cancer Institute: Protocol ID 68529).
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http://dx.doi.org/10.3324/haematol.2011.045229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3291595PMC
March 2012

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Nature 2011 Jul 27;476(7360):298-303. Epub 2011 Jul 27.

Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia V5Z 1L3, Canada.

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.
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http://dx.doi.org/10.1038/nature10351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3210554PMC
July 2011

Xenografts of highly resistant leukemia recapitulate the clonal composition of the leukemogenic compartment.

Blood 2011 Aug 13;118(7):1854-64. Epub 2011 Jun 13.

Division of Oncology and Children's Research Center, University Children's Hospital, University of Zurich, Zurich, Switzerland.

Clonal evolution of the leukemogenic compartment may contribute to alter the therapeutic response in acute lymphoblastic leukemia (ALL). Using xenotransplantation of primary leukemia cells, we evaluated the phenotypic and genetic composition of de novo resistant very high risk precursor B-cell ALL, a subgroup defined by the persistence of minimal residual disease despite intensive chemotherapy. Analysis of copy number alterations (CNAs) showed that the xenografted leukemia, even when reconstituted from 100 cells, remained highly related to the diagnostic sample, with minor changes in CNAs, mostly deletions, emerging in most cases in the first passage into mice. At the single-cell level, the pattern of monoallelic and biallelic deletions of the CDKN2A locus revealed distinct leukemia subpopulations, which were reproducibly tracked in xenografts. In most very high risk ALL cases, the predominant diagnostic clones were reconstituted in xenografts, as shown by multiplex polymerase chain reaction analysis of immunoglobulin and T-cell receptor loci. In other cases, the pattern in CNAs and immunoglobulin and T-cell receptor rearrangement was less concordant in xenografts, suggesting the outgrowth of subclones. These results unequivocally demonstrate the existence of clonally closely related but distinct subsets of leukemia initiating cells in ALL, which has important implications for drug development and preclinical disease modeling.
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http://dx.doi.org/10.1182/blood-2010-11-320309DOI Listing
August 2011

Determining the sub-cellular localization of proteins within Caenorhabditis elegans body wall muscle.

PLoS One 2011 17;6(5):e19937. Epub 2011 May 17.

Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada.

Determining the sub-cellular localization of a protein within a cell is often an essential step towards understanding its function. In Caenorhabditis elegans, the relatively large size of the body wall muscle cells and the exquisite organization of their sarcomeres offer an opportunity to identify the precise position of proteins within cell substructures. Our goal in this study is to generate a comprehensive "localizome" for C. elegans body wall muscle by GFP-tagging proteins expressed in muscle and determining their location within the cell. For this project, we focused on proteins that we know are expressed in muscle and are orthologs or at least homologs of human proteins. To date we have analyzed the expression of about 227 GFP-tagged proteins that show localized expression in the body wall muscle of this nematode (e.g. dense bodies, M-lines, myofilaments, mitochondria, cell membrane, nucleus or nucleolus). For most proteins analyzed in this study no prior data on sub-cellular localization was available. In addition to discrete sub-cellular localization we observe overlapping patterns of localization including the presence of a protein in the dense body and the nucleus, or the dense body and the M-lines. In total we discern more than 14 sub-cellular localization patterns within nematode body wall muscle. The localization of this large set of proteins within a muscle cell will serve as an invaluable resource in our investigation of muscle sarcomere assembly and function.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019937PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3096668PMC
September 2011