Molecular diagnosis of primary mediastinal B cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma

Using current diagnostic criteria, primary mediastinal B cell lymphoma (PMBL) cannot be distinguished from other types of diffuse large B cell lymphoma (DLBCL) reliably. We used gene expression profiling to develop a more precise molecular diagnosis of PMBL. PMBL patients were considerably younger than other DLBCL patients, and their lymphomas frequently involved other thoracic structures but not extrathoracic sites typical of other DLBCLs. PMBL patients had a relatively favorable clinical outcome, with a 5-yr survival rate of 64% compared with 46% for other DLBCL patients. Gene expression profiling strongly supported a relationship between PMBL and Hodgkin lymphoma: over one third of the genes that were more highly expressed in PMBL than in other DLBCLs were also characteristically expressed in Hodgkin lymphoma cells. PDL2, which encodes a regulator of T cell activation, was the gene that best discriminated PMBL from other DLBCLs and was also highly expressed in Hodgkin lymphoma cells. The genomic loci for PDL2 and several neighboring genes were amplified in over half of the PMBLs and in Hodgkin lymphoma cell lines. The molecular diagnosis of PMBL should significantly aid in the development of therapies tailored to this clinically and pathogenetically distinctive subgroup of DLBCL.     

Cernunnos influences human immunoglobulin class switch recombination and may be associated with B cell lymphomagenesis

Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells, with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.     

Shifts in targeting of class switch recombination sites in mice that lack mu switch region tandem repeats or Msh2

The mechanisms that target class switch recombination (CSR) to antibody gene switch (S) regions are unknown. Analyses of switch site locations in wild-type mice and in mice that lack the Smu tandem repeats show shifts indicating that a 4-5-kb DNA domain (bounded upstream by the Imu promoter) is accessible for switching independent of Smu sequences. This CSR-accessible domain is reminiscent of the promoter-defined domains that target somatic hypermutation. Within the 4-5-kb CSR domain, the targeting of S site locations also depends on the Msh2 mismatch repair protein because Msh2-deficient mice show an increased focus of sites to the Smu tandem repeat region. We propose that Msh2 affects S site location because sequences with few activation-induced cytidine deaminase targets generate mostly switch DNA cleavages that require Msh2-directed processing to allow CSR joining.     

Microarray gene expression analysis of fixed archival tissue permits molecular classification and identification of potential therapeutic targets in diffuse large B-cell lymphoma

Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) has a poor prognosis. Novel drugs targeting the constitutively activated NF-κB pathway characteristic of ABC-DLBCL are promising, but evaluation depends on accurate activated B cell-like (ABC)/germinal center B cell-like (GCB) molecular classification. This is traditionally performed on gene microarray expression profiles of fresh biopsies, which are not routinely collected, or by immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue, which lacks reproducibility and classification accuracy. We explored the possibility of using routine archival FFPE tissue for gene microarray applications. We examined Affymetrix HG U133 Plus 2.0 gene expression profiles from paired archival FFPE and fresh-frozen tissues of 40 ABC/GCB-classified DLBCL cases to compare classification accuracy and test the potential for this approach to aid the discovery of therapeutic targets and disease classifiers in DLBCL. Unsupervised hierarchical clustering of unselected present probe sets distinguished ABC/GCB in FFPE with remarkable accuracy, and a Bayesian classifier correctly assigned 32 of 36 cases with >90% probability. Enrichment for NF-κB genes was appropriately seen in ABC-DLBCL FFPE tissues. The top discriminatory genes expressed in FFPE separated cases with high statistical significance and contained novel biology with potential therapeutic insights, warranting further investigation. These results support a growing understanding that archival FFPE tissues can be used in microarray experiments aimed at molecular classification, prognostic biomarker discovery, and molecular exploration of rare diseases.     

Sgamma3 switch sequences function in place of endogenous Sgamma1 to mediate antibody class switching

Immunoglobulin heavy chain (IgH) class switch recombination (CSR) replaces the initially expressed IgH Cmu exons with a set of downstream IgH constant region (C(H)) exons. Individual sets of C(H) exons are flanked upstream by long (1-10-kb) repetitive switch (S) regions, with CSR involving a deletional recombination event between the donor Smu region and a downstream S region. Targeting CSR to specific S regions might be mediated by S region-specific factors. To test the role of endogenous S region sequences in targeting specific CSR events, we generated mutant B cells in which the endogenous 10-kb Sgamma1 region was replaced with wild-type (WT) or synthetic 2-kb Sgamma3 sequences or a synthetic 2-kb Sgamma1 sequence. We found that both the inserted endogenous and synthetic Sgamma3 sequences functioned similarly to a size-matched synthetic Sgamma1 sequence to mediate substantial CSR to IgG1 in mutant B cells activated under conditions that stimulate IgG1 switching in WT B cells. We conclude that Sgamma3 can function similarly to Sgamma1 in mediating endogenous CSR to IgG1. The approach that we have developed will facilitate assays for IgH isotype-specific functions of other endogenous S regions.     

不同免疫表型弥漫大B细胞淋巴瘤的抑癌基因TP53缺失分析

目的探讨不同免疫表型的弥漫大B细胞淋巴瘤(diffuse large B-cell 1ymphoma,DLBCL)抑癌基因TP53缺失情况的差异。方法根据淋巴瘤细胞免疫标志物CD10、Bcl-6及MUM-1的表达情况,将57例DLBCL石蜡包埋组织标本分为GCB(生发中心样B细胞)与non-GCB(非生发中心样B细胞)淋巴瘤两类,用荧光原位杂交(FISH)技术检测GCB与non-GCB类中TP53缺失情况。结果 24例GCB类中有4例(16.7%)TP53缺失,33例non-GCB类中有14例(42.4%)TP53缺失。二者TP53缺失率差异有统计学意义(P<0.05)。结论 non-GCB类患者TP53缺失发生率较高,TP53缺失是non-GCB类预后差的可能原因之一。FISH可以快速、准确、灵敏地检测出TP53的缺失。     

DNA-PKcs and Artemis function in the end-joining phase of immunoglobulin heavy chain class switch recombination

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Artemis are classical nonhomologous DNA end-joining (C-NHEJ) factors required for joining a subset of DNA double-strand breaks (DSB), particularly those requiring end processing. In mature B cells, activation-induced cytidine deaminase (AID) initiates class switch recombination (CSR) by introducing lesions into S regions upstream of two recombining C(H) exons, which are processed into DSBs and rejoined by C-NHEJ to complete CSR. The function of DNA-PKcs in CSR has been controversial with some reports but not others showing that DNA-PKcs-deficient mice are significantly impaired for CSR. Artemis-deficient B cells reportedly undergo CSR at normal levels. Overall, it is still not known whether there are any CSR-associated DSBs that require DNA-PKcs and/or Artemis to be joined. Here, we have used an immunoglobulin (Ig)H locus-specific fluorescent in situ hybridization assay to unequivocally demonstrate that both DNA-PKcs and, unexpectedly, Artemis are necessary for joining a subset of AID-dependent DSBs. In the absence of either factor, B cells activated for CSR frequently generate AID-dependent IgH locus chromosomal breaks and translocations. We also find that under specific activation conditions, DNA-PKcs(-/-) B cells with chromosomal breaks are eliminated or at least prevented from progressing to metaphase via a p53-dependent response.     

Constitutive expression of AID leads to tumorigenesis

Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy.     

Activation-induced deaminase (AID)-directed hypermutation in the immunoglobulin Smu region: implication of AID involvement in a common step of class switch recombination and somatic hypermutation

Somatic hypermutation (SHM) and class switch recombination (CSR) cause distinct genetic alterations at different regions of immunoglobulin genes in B lymphocytes: point mutations in variable regions and large deletions in S regions, respectively. Yet both depend on activation-induced deaminase (AID), the function of which in the two reactions has been an enigma. Here we report that B cell stimulation which induces CSR but not SHM, leads to AID-dependent accumulation of SHM-like point mutations in the switch mu region, uncoupled with CSR. These findings strongly suggest that AID itself or a single molecule generated by RNA editing function of AID may mediate a common step of SHM and CSR, which is likely to be involved in DNA cleavage.     

SELDI-TOF-MS技术在弥漫性大B细胞淋巴瘤诊断中的意义

目的应用表面增强激光解析电离飞行时间质谱（SELDI—TDF-MS）技术从弥漫性大B细胞淋巴瘤（DLBCL）石蜡标本中筛选差异蛋白,并检测生发中心型（GCB型）与非生发中心型（non-GCB型）弥漫性大B细胞淋巴瘤之间的差异蛋白。方法运用免疫组化方法检测41例DLBCL中CD3、CD20、CD10、bcl-6和MUM-1的表达,依据Hans分类方法将DLBCL分为预后有显著差异的两种亚型：GCB型和non—GCB型。选用WCX2芯片及SELDI—TOF-MS技术对这两组亚型的石蜡标本进行蛋白指纹图谱检测并比较其不同。15例淋巴结反应性增生的石蜡组织标本作为对照。用Biomarker Wizard软件对数据进行分析。结果在质荷比位于2～20kDa的范围内,以波峰的信噪比（S／N）〉5为标准,经软件分析发现DLBCL与淋巴结反应性增生之间共有6个蛋白波峰强度值存在明显差异（P〈0．05）。GCB型与non—GCB型DLBCL中共有2个蛋白波峰强度值存在明显差异（P〈0．05）。结论免疫组化与SELDI蛋白芯片技术相结合可检测DLBCL患者的特异性标记物,差异蛋白可能有助于DLBCL的诊断。     

Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma

Rationally designed combinations of targeted therapies for refractory cancers, such as activated B cell–like diffuse large B cell lymphoma (ABC DLBCL), are likely required to achieve potent, durable responses. Here, we used a pharmacoproteomics approach to map the interactome of a tumor-enriched isoform of HSP90 (teHSP90). Specifically, we chemically precipitated teHSP90-client complexes from DLBCL cell lines with the small molecule PU-H71 and found that components of the proximal B cell receptor (BCR) signalosome were enriched within teHSP90 complexes. Functional assays revealed that teHSP90 facilitates BCR signaling dynamics by enabling phosphorylation of key BCR signalosome components, including the kinases SYK and BTK. Consequently, treatment of BCR-dependent ABC DLBCL cells with PU-H71 attenuated BCR signaling, calcium flux, and NF-κB signaling, ultimately leading to growth arrest. Combined exposure of ABC DLBCL cell lines to PU-H71 and ibrutinib, a BCR pathway inhibitor, more potently suppressed BCR signaling than either drug alone. Correspondingly, PU-H71 combined with ibrutinib induced synergistic killing of lymphoma cell lines, primary human lymphoma specimens ex vivo, and lymphoma xenografts in vivo, without notable toxicity. Together, our results demonstrate that a pharmacoproteome-driven rational combination therapy has potential to provide more potent BCR-directed therapy for ABC DLCBL patients.     

DNA repair genes are selectively mutated in diffuse large B cell lymphomas

DNA repair mechanisms are fundamental for B cell development, which relies on the somatic diversification of the immunoglobulin genes by V(D)J recombination, somatic hypermutation, and class switch recombination. Their failure is postulated to promote genomic instability and malignant transformation in B cells. By performing targeted sequencing of 73 key DNA repair genes in 29 B cell lymphoma samples, somatic and germline mutations were identified in various DNA repair pathways, mainly in diffuse large B cell lymphomas (DLBCLs). Mutations in mismatch repair genes (EXO1, MSH2, and MSH6) were associated with microsatellite instability, increased number of somatic insertions/deletions, and altered mutation signatures in tumors. Somatic mutations in nonhomologous end-joining (NHEJ) genes (DCLRE1C/ARTEMIS, PRKDC/DNA-PKcs, XRCC5/KU80, and XRCC6/KU70) were identified in four DLBCL tumors and cytogenetic analyses revealed that translocations involving the immunoglobulin-heavy chain locus occurred exclusively in NHEJ-mutated samples. The novel mutation targets, CHEK2 and PARP1, were further screened in expanded DLBCL cohorts, and somatic as well as novel and rare germline mutations were identified in 8 and 5% of analyzed tumors, respectively. By correlating defects in a subset of DNA damage response and repair genes with genomic instability events in tumors, we propose that these genes play a role in DLBCL lymphomagenesis.Normal lymphocyte development and function relies on the successful rearrangement and modification of antigen receptor genes. Diversity of the B cell receptor is largely provided by V(D)J recombination where the variable (V), diversity (D), and joining (J) segments of the immunoglobulin (IG) loci are joined in a combinatorial manner (Jung et al., 2006). After antigen experience, the IG loci are further modified by somatic hypermutation (SHM) and class switch recombination (CSR). The recombination-activating proteins 1 and 2 (RAG1 and RAG2) introduce double-strand breaks (DSBs) at recombination signal sequences located around the V, D, and J genes during V(D)J recombination, whereas activation-induced cytidine deaminase (AID) initiates SHM and CSR by deaminating cytosines to uracils at the V and switch (S) regions of the IG loci (Jung et al., 2006; Di Noia and Neuberger, 2007).A myriad of DNA damage response (DDR) and repair proteins mediate and regulate IG diversification processes. AID activity provokes guanosine/uracil mismatches that are processed by proteins of the base-excision repair (BER) pathway (UNG, APEX1) and mismatch repair (MMR) pathway (MSH2/MSH6, MLH1/PMS2, and EXO1; Di Noia and Neuberger, 2007; Stavnezer et al., 2010). While in the context of CSR such mismatches lead to the generation of DNA DSBs, during SHM, AID activity preferentially results in the establishment of point mutations, although small duplications and deletions may also occur. The resolution of DSBs in V(D)J recombination and CSR is primarily mediated by the nonhomologous end-joining (NHEJ) pathway that becomes activated by DDR proteins such as ATM and Nibrin (NBN; Kotnis et al., 2009). The x-ray repair cross-complementing proteins 4 (XRCC4), XRCC5 (Ku80), and XRCC6 (Ku70) and DNA ligase 4, Artemis, DNA-PKcs, and Cernunnos (XLF) proteins are considered to be the core members of NHEJ (Lieber, 2010).Most B cell neoplasms are thought to originate from antigen-experienced B cells, as tumor cells display SHM at the IG V genes (Klein and Dalla-Favera, 2008). Furthermore, a role for IG diversification mechanisms in the propagation of genomic instability in mature B cell lymphomas is supported by numerous observations. Non-IG genes, including proto-oncogenes such as MYC, BCL6, PIM1, RHOH, or PAX5 are often targeted by SHM, especially in diffuse large B cell lymphomas (DLBCLs; Pasqualucci et al., 2001), one of the most common and aggressive mature B cell lymphoma subtypes. Chromosomal translocations involving the IG loci, with breakpoints in S regions and SHM targets, are also a hallmark of mature B cell lymphomas (Küppers and Dalla-Favera, 2001; Lenz et al., 2007). Furthermore, AID has been shown to be essential for the occurrence of c-myc/IgH translocations and oncogene-driven induction of germinal center–derived lymphomas in mice (Ramiro et al., 2004; Pasqualucci et al., 2008). Of note, the t(14;18) translocation, involving the IGH and the BCL2 loci, characteristic of follicular lymphomas (FLs), is considered to be derived from defective V(D)J recombination processes (Küppers and Dalla-Favera, 2001).Despite the crucial role of DDR and repair proteins during antibody diversification processes, there is lack of evidence that supports their direct involvement in the propagation of genomic instability in human B cell lymphomas. In contrast, individuals with biallelic germline mutations in some of the DNA repair genes that encode proteins involved in IG diversification processes often display an increased risk for development of lymphoid malignancies in addition to immunodeficiency (de Miranda et al., 2011). In this study, we systematically analyzed the coding regions of key DDR and repair genes that have been associated, or could potentially be associated, with IG gene diversification processes in a set of mature B cell lymphomas, with a focus on DLBCL. The defects in a subset of DDR and repair genes identified here, and their association with genomic instability phenotypes, support their role in the tumorigenesis of DLBCL.     

鼻咽部弥漫性大B细胞淋巴瘤临床病理分析

目的探讨鼻咽部弥漫性大B细胞淋巴瘤（DLBCL）的临床病理特征以提高诊断水平。方法回顾性分析12例鼻咽部DLBCL患者临床病理资料,所有病例均进行CD20、CD3、CD10、BCL6、MUM．1、Ki-67免疫表型测定,部分加做AE1／3、CD30、ALK、CD5和CyclinD1。结果12例鼻咽部DLBCL均为原发,男8例,女4例,年龄34—67岁,平均48岁;5例临床误诊为鼻咽癌。生发中心细胞型7例,活化的B细胞型5例。结论鼻咽部DLBCL极易误诊为鼻咽癌,形态学、免疫表型及临床表现三者结合可确诊。     

DNA polymerase eta is involved in hypermutation occurring during immunoglobulin class switch recombination

Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase eta (pol eta), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol eta is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes.     

鼻咽部弥漫性大B细胞淋巴瘤临床病理分析

目的 探讨鼻咽部弥漫性大B细胞淋巴瘤(DLBCL)的临床病理特征以提高诊断水平.方法 回顾性分析12例鼻咽部DLBCL患者临床病理资料,所有病例均进行CD20、CD3、CD10、BCL6、MUM-1、Ki-67免疫表型测定,部分加做AE1/3、CD30、ALK、CD5和CyclinD1.结果 12例鼻咽部DLBCL均为原发,男8例,女4例,年龄34～67岁,平均48岁;5例临床误诊为鼻咽癌.生发中心细胞型7例,活化的B细胞型5例.结论 鼻咽部DLBCL极易误诊为鼻咽癌,形态学、免疫表型及临床表现三者结合可确诊.