RESUMO
Fluorescence in situ hybridization (FISH) using break-apart probes is recommended for identifying high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). Unbalanced MYC break-apart patterns, where the red or green signal is lost, are commonly reported as an equivocal result by clinical laboratories. In a cohort of 297 HGBCL-DH-BCL2, 13% of tumors had unbalanced MYC break-apart patterns with loss of red (LR: 2%) or green (LG: 11%) signal. To determine the significance of these patterns, MYC rearrangements were characterized by sequencing in 130 HGBCL-DH-BCL2, including 3 LR and 14 LG tumors. A MYC rearrangement was identified for 71% of tumors with LR or LG patterns, with the majority involving immunoglobulin loci or other recurrent MYC rearrangement partners. The architecture of these rearrangements consistently preserved the rearranged MYC allele, with the MYC gene predicted to be on the derivative chromosome containing the signal that is still present in nearly all cases. MYC protein expression, MYC mRNA expression, and the proportion of tumors expressing the dark zone signature was not significantly different between balanced and unbalanced groups. These results support a recommendation that unbalanced MYC break-apart FISH patterns be reported as positive for MYC rearrangement in the context of diagnosing HGBCL-DH-BCL2.
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Recent genetic and molecular classification of DLBCL has advanced our knowledge of disease biology, yet were not designed to predict early events and guide anticipatory selection of novel therapies. To address this unmet need, we used an integrative multiomic approach to identify a signature at diagnosis that will identify DLBCL at high risk of early clinical failure. Tumor biopsies from 444 newly diagnosed DLBCL were analyzed by WES and RNAseq. A combination of weighted gene correlation network analysis and differential gene expression analysis was used to identify a signature associated with high risk of early clinical failure independent of IPI and COO. Further analysis revealed the signature was associated with metabolic reprogramming and identified cases with a depleted immune microenvironment. Finally, WES data was integrated into the signature and we found that inclusion of ARID1A mutations resulted in identification of 45% of cases with an early clinical failure which was validated in external DLBCL cohorts. This novel and integrative approach is the first to identify a signature at diagnosis, in a real-world cohort of DLBCL, that identifies patients at high risk for early clinical failure and may have significant implications for design of therapeutic options.
Assuntos
Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/diagnóstico , Masculino , Feminino , Perfilação da Expressão Gênica , Pessoa de Meia-Idade , Transcriptoma , Mutação , Regulação Neoplásica da Expressão Gênica , Fatores de Transcrição/genética , Biomarcadores Tumorais/genética , Idoso , Prognóstico , Microambiente Tumoral , Sequenciamento do Exoma , Adulto , Proteínas de Ligação a DNA/genética , Falha de TratamentoRESUMO
Proteome coverage and accurate protein quantification are both important for evaluating biological systems; however, compromises between quantification, coverage, and mass spectrometry (MS) resources are often necessary. Consequently, experimental parameters that impact coverage and quantification must be adjusted, depending on experimental goals. Among these parameters is offline prefractionation, which is utilized in MS-based proteomics to decrease sample complexity resulting in higher overall proteome coverage upon MS analysis. Prefractionation leads to increases in required MS analysis time, although this is often mitigated by isobaric labeling using tandem-mass tags (TMT), which allow samples to be multiplexed. Here we evaluate common prefractionation schemes, TMT variants, and MS acquisition methods and their impact on protein quantification and coverage. Furthermore, we provide recommendations for experimental design depending on the experimental goals.
Assuntos
Proteoma , Proteômica , Espectrometria de Massas em Tandem , Proteômica/métodos , Proteômica/normas , Espectrometria de Massas em Tandem/métodos , Proteoma/análise , Humanos , Fracionamento Químico/métodos , Coloração e Rotulagem/métodosRESUMO
ABSTRACT: Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit"; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.
Assuntos
Rearranjo Gênico , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-myc , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Linfoma de Células B/genética , Linfoma de Células B/patologia , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.
Assuntos
Linfoma Difuso de Grandes Células B , Microambiente Tumoral , Humanos , Microambiente Tumoral/genética , Interleucina-4/genética , Interleucina-4/metabolismo , Interleucina-4/farmacologia , Recidiva Local de Neoplasia , Linfoma Difuso de Grandes Células B/patologia , Mutação , Fator de Transcrição STAT6/genética , Fator de Transcrição STAT6/metabolismoRESUMO
Diffuse large B-cell lymphoma (DLBCL) is heterogeneous both in clinical outcomes and the underlying disease biology. Over the last 2 decades, several different approaches for dissecting biological heterogeneity have emerged. Gene expression profiling (GEP) stratifies DLBCL into 3 broad groups (ABC, GCB, and DZsig/MHG), each with parallels to different normal mature B cell developmental states and prognostic implications. More recently, several different genomic approaches have been developed to categorize DLBCL based on the co-occurrence of tumor somatic mutations, identifying more granular biologically unified subgroups that complement GEP-based approaches. We review the molecular approaches and clinical evidence supporting the stratification of DLBCL patients based on tumor biology. By offering a platform for subtype-guided therapy, these divisions remain a promising avenue for improving patient outcomes, especially in subgroups with inferior outcomes with current standard-of-care therapy.
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Linfoma Difuso de Grandes Células B , Humanos , Linfoma Difuso de Grandes Células B/terapia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Perfilação da Expressão Gênica , Prognóstico , GenômicaRESUMO
Mantle cell lymphoma (MCL) is a lethal hematological malignancy with a median survival of 4 years. Its lethality is mainly attributed to a limited understanding of clinical tumor progression and resistance to current therapeutic regimes. Intrinsic, prolonged drug treatment and tumor-microenvironment (TME) facilitated factors impart pro-tumorigenic and drug-insensitivity properties to MCL cells. Hence, elucidating neoteric pharmacotherapeutic molecular targets involved in MCL progression utilizing a global "unified" analysis for improved disease prevention is an earnest need. Using integrated transcriptomic analyses in MCL patients, we identified a Fibroblast Growth Factor Receptor-1 (FGFR1), and analyses of MCL patient samples showed that high FGFR1 expression was associated with shorter overall survival in MCL patient cohorts. Functional studies using pharmacological intervention and loss of function identify a novel MYC-EZH2-CDKN1C axis-driven proliferation in MCL. Further, pharmacological targeting with erdafitinib, a selective small molecule targeting FGFRs, induced cell-cycle arrest and cell death in-vitro, inhibited tumor progression, and improved overall survival in-vivo. We performed extensive pre-clinical assessments in multiple in-vivo model systems to confirm the therapeutic potential of erdafitinib in MCL and demonstrated FGFR1 as a viable therapeutic target in MCL.
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Linfoma de Célula do Manto , Adulto , Humanos , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Linfoma de Célula do Manto/tratamento farmacológico , Linfoma de Célula do Manto/genética , Linfoma de Célula do Manto/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Microambiente Tumoral/genéticaRESUMO
PURPOSE: Diffuse large B-cell lymphoma (DLBCL) is cured in more than 60% of patients, but outcomes remain poor for patients experiencing disease progression or relapse (refractory or relapsed DLBCL [rrDLBCL]), particularly if these events occur early. Although previous studies examining cohorts of rrDLBCL have identified features that are enriched at relapse, few have directly compared serial biopsies to uncover biological and evolutionary dynamics driving rrDLBCL. Here, we sought to confirm the relationship between relapse timing and outcomes after second-line (immuno)chemotherapy and determine the evolutionary dynamics that underpin that relationship. PATIENTS AND METHODS: Outcomes were examined in a population-based cohort of 221 patients with DLBCL who experienced progression/relapse after frontline treatment and were treated with second-line (immuno)chemotherapy with an intention-to-treat with autologous stem-cell transplantation (ASCT). Serial DLBCL biopsies from a partially overlapping cohort of 129 patients underwent molecular characterization, including whole-genome or whole-exome sequencing in 73 patients. RESULTS: Outcomes to second-line therapy and ASCT are superior for late relapse (>2 years postdiagnosis) versus primary refractory (<9 months) or early relapse (9-24 months). Diagnostic and relapse biopsies were mostly concordant for cell-of-origin classification and genetics-based subgroup. Despite this concordance, the number of mutations exclusive to each biopsy increased with time since diagnosis, and late relapses shared few mutations with their diagnostic counterpart, demonstrating a branching evolution pattern. In patients with highly divergent tumors, many of the same genes acquired new mutations independently in each tumor, suggesting that the earliest mutations in a shared precursor cell constrain tumor evolution toward the same genetics-based subgroups at both diagnosis and relapse. CONCLUSION: These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease and have implications for optimal patient management.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Linfoma Difuso de Grandes Células B , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Linfoma Difuso de Grandes Células B/terapia , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Doença Crônica , Transplante Autólogo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
PURPOSE: 60-70% of newly diagnosed diffuse large B-cell lymphoma (DLBCL) patients avoid events within 24 months of diagnosis (EFS24) and the remainder have poor outcomes. Recent genetic and molecular classification of DLBCL has advanced our knowledge of disease biology, yet were not designed to predict early events and guide anticipatory selection of novel therapies. To address this unmet need, we used an integrative multiomic approach to identify a signature at diagnosis that will identify DLBCL at high risk of early clinical failure. PATIENTS AND METHODS: Tumor biopsies from 444 newly diagnosed DLBCL were analyzed by WES and RNAseq. A combination of weighted gene correlation network analysis and differential gene expression analysis followed by integration with clinical and genomic data was used to identify a multiomic signature associated with high risk of early clinical failure. RESULTS: Current DLBCL classifiers are unable to discriminate cases who fail EFS24. We identified a high risk RNA signature that had a hazard ratio (HR, 18.46 [95% CI 6.51-52.31] P < .001) in a univariate model, which did not attenuate after adjustment for age, IPI and COO (HR, 20.8 [95% CI, 7.14-61.09] P < .001). Further analysis revealed the signature was associated with metabolic reprogramming and a depleted immune microenvironment. Finally, WES data was integrated into the signature and we found that inclusion of ARID1A mutations resulted in identification of 45% of cases with an early clinical failure which was validated in external DLBCL cohorts. CONCLUSION: This novel and integrative approach is the first to identify a signature at diagnosis that will identify DLBCL at high risk for early clinical failure and may have significant implications for design of therapeutic options.
Assuntos
Linfoma Difuso de Grandes Células B , RNA Longo não Codificante , Humanos , Transcriptoma , RNA Longo não Codificante/genética , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Perfilação da Expressão Gênica , Resultado do TratamentoRESUMO
Follicular lymphoma (FL) accounts for â¼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.
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Linfoma Folicular , Linfoma Difuso de Grandes Células B , Humanos , Linfoma Folicular/patologia , Mutação , Linfoma Difuso de Grandes Células B/genética , Linfoma Difuso de Grandes Células B/patologiaRESUMO
Diffuse large B-cell lymphoma (DLBCL) is cured in over 60% of patients, but outcomes are poor for patients with relapsed or refractory disease (rrDLBCL). Here, we performed whole genome/exome sequencing (WGS/WES) on tumors from 73 serially-biopsied patients with rrDLBCL. Based on the observation that outcomes to salvage therapy/autologous stem cell transplantation are related to time-to-relapse, we stratified patients into groups according to relapse timing to explore the relationship to genetic divergence and sensitivity to salvage immunochemotherapy. The degree of mutational divergence increased with time between biopsies, yet tumor pairs were mostly concordant for cell-of-origin, oncogene rearrangement status and genetics-based subgroup. In patients with highly divergent tumors, several genes acquired exclusive mutations independently in each tumor, which, along with concordance of genetics-based subgroups, suggests that the earliest mutations in a shared precursor cell constrain tumor evolution. These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naâØve disease.
RESUMO
Heterogeneous nuclear ribonucleoproteins (hnRNPs) are among the most abundantly expressed RNA binding proteins in the cell and play major roles in all facets of RNA metabolism. hnRNPs are increasingly appreciated as essential for mammalian B cell development by regulating the carefully ordered expression of specific genes. Due to this tight regulation of the hnRNP-RNA network, it is no surprise that a growing number of genes encoding hnRNPs have been causally associated with the onset or progression of many cancers, including B cell neoplasms. Here we discuss our current understanding of hnRNP-driven regulation in normal, perturbed, and malignant B cells, and the most recent and emerging therapeutic innovations aimed at targeting the hnRNP-RNA network in lymphoma.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas , Ribonucleoproteínas , Animais , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Mamíferos/genética , Mamíferos/metabolismoRESUMO
Targeted and semitargeted mass spectrometry-based approaches are reliable methods to consistently detect and quantify low abundance proteins including proteins of clinical significance. Despite their potential, the development of targeted and semitargeted assays is time-consuming and often requires the purchase of costly libraries of synthetic peptides. To improve the efficiency of this rate-limiting step, we developed PeptideRanger, a tool to identify peptides from protein of interest with physiochemical properties that make them more likely to be suitable for mass spectrometry analysis. PeptideRanger is a flexible, extensively annotated, and intuitive R package that uses a random forest model trained on a diverse data set of thousands of MS experiments spanning a variety of sample types profiled with different chromatography setups and instruments. To support a variety of applications and to leverage rapidly growing public MS databases, PeptideRanger can readily be retrained with experiment-specific data sets and customized to prioritize and filter peptides based on selected properties.
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Peptídeos , Proteômica , Proteômica/métodos , Peptídeos/análise , Espectrometria de Massas/métodos , ProteínasRESUMO
Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.
Assuntos
Linfoma Difuso de Grandes Células B , Proteínas Proto-Oncogênicas c-myc , Humanos , Hibridização in Situ Fluorescente , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/epidemiologia , Linfoma Difuso de Grandes Células B/genética , PrognósticoAssuntos
Linfoma de Burkitt , Humanos , Linfoma de Burkitt/genética , Mutação , Translocação GenéticaRESUMO
Follicular lymphoma (FL) is an indolent cancer of mature B-cells but with ongoing risk of transformation to more aggressive histology over time. Recurrent mutations associated with transformation have been identified; however, prognostic features that can be discerned at diagnosis could be clinically useful. We present here comprehensive profiling of both tumor and immune compartments in 155 diagnostic FL biopsies at single-cell resolution by mass cytometry. This revealed a diversity of phenotypes but included two recurrent patterns, one which closely resembles germinal center B-cells (GCB) and another which appears more related to memory B-cells (MB). GCB-type tumors are enriched for EZH2, TNFRSF14, and MEF2B mutations, while MB-type tumors contain increased follicular helper T-cells. MB-type and intratumoral phenotypic diversity are independently associated with increased risk of transformation, supporting biological relevance of these features. Notably, a reduced 26-marker panel retains sufficient information to allow phenotypic profiling of future cohorts by conventional flow cytometry.
Assuntos
Linfoma Folicular , Humanos , Linfoma Folicular/genética , Células B de Memória , Centro Germinativo , Linfócitos B , MutaçãoRESUMO
Exome and genome sequencing has facilitated the identification of hundreds of genes and other regions that are recurrently mutated in hematologic neoplasms. The data sets from these studies theoretically provide opportunities. Quality differences between data sets can confound secondary analyses. We explore the consequences of these on the conclusions from some recent studies of B-cell lymphomas. We highlight the need for a minimum reporting standard to increase transparency in genomic research.