Kui Nie

Epigenomic evolution in diffuse large B-cell lymphomas.

The contribution of epigenomic alterations to tumour progression and relapse is not well characterized. Here we characterize an association between disease progression and DNA methylation in diffuse large B-cell lymphoma (DLBCL). By profiling genome-wide DNA methylation at single-base pair resolution in thirteen DLBCL diagnosis–relapse sample pairs, we show that DLBCL patients exhibit heterogeneous evolution of tumour methylomes during relapse. We identify differentially methylated regulatory elements and determine a relapse-associated methylation signature converging on key pathways such as transforming growth factor-β (TGF-β) receptor activity. We also observe decreased intra-tumour methylation heterogeneity from diagnosis to relapsed tumour samples. Relapse-free patients display lower intra-tumour methylation heterogeneity at diagnosis compared with relapsed patients in an independent validation cohort. Furthermore, intra-tumour methylation heterogeneity is predictive of time to relapse. Therefore, we propose that epigenomic heterogeneity may support or drive the relapse phenotype and can be used to predict DLBCL relapse.

Deep Sequencing Reveals Clonal Evolution Patterns and Mutation Events Associated With Relapse In B Cell Lymphomas

BackgroundMolecular mechanisms associated with frequent relapse of diffuse large B-cell lymphoma (DLBCL) are poorly defined. It is especially unclear how primary tumor clonal heterogeneity contributes to relapse. Here, we explore unique features of B-cell lymphomas - VDJ recombination and somatic hypermutation - to address this question.ResultsWe performed high-throughput sequencing of rearranged VDJ junctions in 14 pairs of matched diagnosis-relapse tumors, among which 7 pairs were further characterized by exome sequencing. We identify two distinctive modes of clonal evolution of DLBCL relapse: an early-divergent mode in which clonally related diagnosis and relapse tumors diverged early and developed in parallel; and a late-divergent mode in which relapse tumors developed directly from diagnosis tumors with minor divergence. By examining mutation patterns in the context of phylogenetic information provided by VDJ junctions, we identified mutations in epigenetic modifiers such as KMT2D as potential early driving events in lymphomagenesis and immune escape alterations as relapse-associated events.ConclusionsAltogether, our study for the first time provides important evidence that DLBCL relapse may result from multiple, distinct tumor evolutionary mechanisms, providing rationale for therapies for each mechanism. Moreover, this study highlights the urgent need to understand the driving roles of epigenetic modifier mutations in lymphomagenesis, and immune surveillance factor genetic lesions in relapse.

Epigenetic Down-Regulation of the Tumor Suppressor Gene PRDM1/Blimp-1 in Diffuse Large B Cell Lymphomas: A Potential Role of the MicroRNA Let-7

PRDM1/Blimp-1, a master regulator for B cell terminal differentiation, is a putative tumor suppressor in diffuse large B cell lymphomas (DLBCL). Inactivating mutations of PRDM1 have been previously identified in a subset of nongerminal center B cell-like (GCB) DLBCL. We investigated the presence of alternative mechanisms of down-regulating PRDM1 in a cohort of 25 primary DLBCL and six DLBCL cell lines. While some DLBCL, predominantly the GCB-type, showed low levels of both PRDM1alpha mRNA and protein, presumably as a result of direct transcription repression, discordant expressions between the two were identified in a subset of DLBCL without PRDM1 mutations, the primarily non-GCB type, consistent with translational down-regulation. This subset of DLBCL exhibits relatively high PRDM1alpha mRNA levels but low levels of PRDM1. Data obtained from expression analysis, luciferase reporter assays, and transfection experiments support a role of targeting of PRDM1 by microRNA let-7 family in mediating this down-regulation. Let-7, in particular let-7b, is overexpressed in DLBCL relative to normal GCB cells, suggesting that it is deregulated. Thus, abnormal epigenetic down-regulation of PRDM1 by let-7 and other microRNAs may represent an alternative mechanism of reducing normal PRDM1 function in a subset of DLBCL with relatively high PRDM1alpha mRNA expression and unmutated PRDM1. These findings provide further evidence for an important role of impairment of terminal B cell differentiation in DLBCL pathogenesis.

Transformation of Follicular Lymphoma to Plasmablastic Lymphoma With c-myc Gene Rearrangement

Follicular lymphoma (FL) is an indolent lymphoma that transforms to high-grade lymphoma, mostly diffuse large B-cell lymphoma, in about a third of patients. We present the first report of a case of FL that transformed to plasmablastic lymphoma (PBL). Clonal transformation of the FL to PBL was evidenced by identical IGH/BCL2 gene rearrangements and VDJ gene usage in rearranged IGH genes. IGH/ BCL2 translocation was retained in the PBL, which also acquired c-myc gene rearrangement. Genealogic analysis based on somatic hypermutation of the rearranged IGH genes of both FL and PBL suggests that transformation of the FL to PBL occurred most likely by divergent evolution from a common progenitor cell rather than direct evolution from the FL clone. Our study of this unusual case expands the histologic spectrum of FL transformation and increases our understanding of the pathogenetic mechanisms of transformation of indolent lymphomas to aggressive lymphomas.

EBV-miR-BHRF1-2 targets PRDM1/Blimp1: potential role in EBV lymphomagenesis

PRDM1/Blimp1, a master regulator of B-cell terminal differentiation, has been identified as a tumor suppressor gene in aggressive lymphomas, including diffuse large B-cell lymphoma (DLBCL). It has been shown in DLBCL and Hodgkin lymphoma that PRDM1 is downregulated by cellular microRNAs. In this study, we identify the Epstein–Barr virus (EBV) microRNA (miRNA), EBV-miR-BHRF1-2, as a viral miRNA regulator of PRDM1. EBV-miR-BHRF1-2 repressed luciferase reporter activity by specific interaction with the seed region within the PRDM1 3’ untranslated region. EBV-miR-BHRF1-2 inhibition upregulated PRDM1 protein expression in lymphoblastoid cell lines (LCL), supporting a role of miR-BHRF1-2 in PRDM1 downregulation in vivo. Discordance of PRDM1 messenger RNA and protein expressions is associated with high EBV-miR-BHRF1-2 levels in LCLs and primary post-transplant EBV-positive DLBCL. Enforced expression of PRDM1-induced apoptosis and cell cycle arrest in LCL cells. Inhibition of EBV-miR-BHRF1-2 negatively regulates cell cycle and decreases expression of SCARNA20, a small nucleolar RNA that is also downregulated by PRDM1 overexpression. The interaction between EBV-miR-BHRF1-2 and PRDM1 may be one of the mechanisms by which EBV-miR-BHRF1-2 promotes EBV lymphomagenesis. Our results support the potential of EBV-miR-BHRF1-2 as a therapeutic target in EBV-associated lymphoma.

BIC is processed efficiently to microRNA-155 in Burkitt lymphoma cells

MicroRNAs are short single-stranded RNAs derived from precursors through processing by RNase III enzymes Drosha and Dicer. They function as gene regulators in a variety of physiological processes. Abnormal expression of microRNA is highly implicated in oncogenesis and can be caused by aberrations in levels of transcription of primary microRNA precursors, as well as post-transcriptional alterations. MiR-155 is one of the most well-known oncogenic microRNA. MiR-155 overexpression has been documented in a number of lymphoid neoplasms, such as chronic lymphocytic leukemia, diffuse large B-cell lymphomas, Hodgkin’s lymphoma and primary mediastinal large B-cell lymphoma. However, BIC (the precursor for miR-155) and miR-155 were not detected or detected at very low levels in adult primary Burkitt lymphoma (BL) tissues. A defect in BIC processing has been postulated in BL cells. In this study, we evaluated the capacity of BIC to be processed into mature miR-155 by transient transfection of BL cell line Ramos and expression analysis of BIC and miR-155 in primary BL cases. EBV(-) BL cell line Ramos and human embryonic kidney 293 cells were transiently transfected with pcDNA3 empty vector, pcDNA3.BIC.ex3 or pcDNA.BIC.fl. The construction of pcDNA3.BIC.ex3 has been described previously. It carries the short form of BIC exon 3, where pre-miR-155 resides. pcDNA.BIC.fl was obtained by subcloning the full-length BIC cDNA (RZPD, Berlin, Germany) in pcDNA3. Ramos cells transfected with empty vector harbors only a low level of miR155, approximately 10% that of the prototypic activated B-cell type-DLBCL cell line OCI-Ly3, which harbors high levels of BIC and miR-155. Transient transfection with the miR-155 expression plasmids in Ramos cells resulted in 420-fold increase in miR-155 (Figure 1a). Similarly, transient transfection of 293 cells increased miR-155 levels in these cells by 430-fold. We also looked at the levels of BIC in these transfectants by quantitative real-time PCR using primers and probe specific to exon 3 on cDNA reverse-transcribed from total RNA that is free of genomic

Hypermethylation of the tumor suppressor gene PRDM1/Blimp-1 supports a pathogenetic role in EBV-positive Burkitt lymphoma

PRDM1/Blimp-1 is a tumor suppressor gene in the activated B-cell subtype of diffuse large B-cell lymphomas. Its inactivation contributes to pathogenesis in this setting by impairing terminal B-cell differentiation induced by constitutive nuclear factor-κB activation. The role of PRDM1 in Burkitt lymphoma (BL) lymphomagenesis is not known. Here we identified hypermethylation of the promoter region and exon 1 of PRDM1 in all six Epstein–Barr virus (EBV)-positive BL cell lines and 12 of 23 (52%) primary EBV-positive BL or BL-related cases examined, but in none of the EBV-negative BL cell lines or primary tumors that we assessed, implying a tumor suppressor role for PRDM1 specifically in EBV-associated BL. A direct induction of PRDM1 hypermethylation by EBV is unlikely, as PRDM1 hypermethylation was not observed in EBV-immortalized B lymphoblastoid cell lines. Treatment of EBV-positive BL cells with 5′ azacytidine resulted in PRDM1 induction associated with PRDM1 demethylation, consistent with transcriptional silencing of PRDM1 as a result of DNA methylation. Overexpression of PRDM1 in EBV-positive BL cell lines resulted in cell cycle arrest. Our results expand the spectrum of lymphoid malignancies in which PRDM1 may have a tumor suppressor role and identify an epigenetic event that likely contributes to the pathogenesis of BL.

Composite Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma and Follicular Lymphoma Are Biclonal Lymphomas A Report of Two Cases

Composite lymphomas (CLs) consisting of 2 indolent B-cell lymphomas are rare. We present 2 CL cases composed of chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), each with unique clinicopathologic features. In the first case, the FL was negative for IGH-BCL2 and harbored a novel IGH-associated translocation; in the second case, the CL manifested in the skin. The individual components in both CLs were derived from different B-cell clones. This is the first complete characterization, including molecular analysis, of CLs composed of leukemic CLL and FL and the first report of a cutaneous CL derived from 2 low-grade B cell lymphomas. Our results provide additional supporting evidence that CLs of indolent B-cell lymphomas are biclonal and suggest that they are pathogenetically different from CLs composed of a low-grade B-cell lymphoma and an aggressive B-cell lymphoma or Hodgkin lymphoma, which are usually clonally related.

Significance of PRDM1beta expression as a prognostic marker in diffuse large B-cell lymphomas.

To the editor: PRDM1β is a functionally impaired isoform of PRDM1,[1][1] the master regulator of plasma cell differentiation.[2][2] The paper by Liu et al[3][3] suggested that PRDM1 β expression correlates with chemotherapy resistance in patients with the non–germinal center B-cell (GCB)–type

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma.

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding the clonal composition changes that occur within a tumor in response to certain micro-environment or treatments may lead to the design of more sophisticated and effective approaches to eradicate tumor cells. However, tracking tumor clonal sub-populations has been challenging due to the lack of distinguishable markers. To address this problem, a VDJ-seq protocol was created to trace the clonal evolution patterns of diffuse large B cell lymphoma (DLBCL) relapse by exploiting VDJ recombination and somatic hypermutation (SHM), two unique features of B cell lymphomas. In this protocol, Next-Generation sequencing (NGS) libraries with indexing potential were constructed from amplified rearranged immunoglobulin heavy chain (IgH) VDJ region from pairs of primary diagnosis and relapse DLBCL samples. On average more than half million VDJ sequences per sample were obtained after sequencing, which contain both VDJ rearrangement and SHM information. In addition, customized bioinformatics pipelines were developed to fully utilize sequence information for the characterization of IgH-VDJ repertoire within these samples. Furthermore, the pipeline allows the reconstruction and comparison of the clonal architecture of individual tumors, which enables the examination of the clonal heterogeneity within the diagnosis tumors and deduction of clonal evolution patterns between diagnosis and relapse tumor pairs. When applying this analysis to several diagnosis-relapse pairs, we uncovered key evidence that multiple distinctive tumor evolutionary patterns could lead to DLBCL relapse. Additionally, this approach can be expanded into other clinical aspects, such as identification of minimal residual disease, monitoring relapse progress and treatment response, and investigation of immune repertoires in non-lymphoma contexts.