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MicroRNAs (miRNAs) are small non coding RNAs responsible for posttranscriptional regulation of gene expression. Even though almost 2000 precursors have been described so far, additional miRNAs are still being discovered in normal as well as malignant cells. Alike protein coding genes, miRNAs may acquire oncogenic properties in consequence of altered expression or presence of gain or loss of function mutations. In this study we mined datasets from miRNA expression profiling (miRNA-seq) of 7 classic Hodgkin Lymphoma (cHL) cell lines, 10 non-Hodgkin lymphoma (NHL) cell lines and 56 samples of germinal center derived B-cell lymphomas. Our aim was to discover potential novel cHL oncomiRs not reported in miRBase (release 22.1) and expressed in cHL cell lines but no other B-cell lymphomas. We identified six such miRNA candidates in cHL cell lines and verified the expression of two of them encoded at chr2:212678788-212678849 and chr5:168090507-168090561 (GRCh38). Interestingly, we showed that one of the validated miRNAs (located in an intron of the TENM2 gene) is expressed together with its host gene. TENM2 is characterized by hypomethylation and open chromatin around its TSS in cHL cell lines in contrast to NHL cell lines and germinal centre B-cells respectively. It indicates an epigenetic mechanism responsible for aberrant expression of both, the TENM2 gene and the novel miRNA in cHL cell lines. Despite the GO analysis performed with the input of the in silico predicted novel miRNA target genes did not reveal ontologies typically associated with cHL pathogenesis, it pointed to several interesting candidates involved in i.e. lymphopoiesis. These include the lymphoma related BCL11A gene, the IKZF2 gene involved in lymphocyte development or the transcription initiator GTF2H1., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Ustaszewski et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

Background: Germinal center-derived B cell lymphomas are tumors of the lymphoid tissues representing one of the most heterogeneous malignancies. Here we characterize the variety of transcriptomic phenotypes of this disease based on 873 biopsy specimens collected in the German Cancer Aid MMML (Molecular Mechanisms in Malignant Lymphoma) consortium. They include diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), Burkitt's lymphoma, mixed FL/DLBCL lymphomas, primary mediastinal large B cell lymphoma, multiple myeloma, IRF4-rearranged large cell lymphoma, MYC-negative Burkitt-like lymphoma with chr. 11q aberration and mantle cell lymphoma., Methods: We apply self-organizing map (SOM) machine learning to microarray-derived expression data to generate a holistic view on the transcriptome landscape of lymphomas, to describe the multidimensional nature of gene regulation and to pursue a modular view on co-expression. Expression data were complemented by pathological, genetic and clinical characteristics., Results: We present a transcriptome map of B cell lymphomas that allows visual comparison between the SOM portraits of different lymphoma strata and individual cases. It decomposes into one dozen modules of co-expressed genes related to different functional categories, to genetic defects and to the pathogenesis of lymphomas. On a molecular level, this disease rather forms a continuum of expression states than clearly separated phenotypes. We introduced the concept of combinatorial pattern types (PATs) that stratifies the lymphomas into nine PAT groups and, on a coarser level, into five prominent cancer hallmark types with proliferation, inflammation and stroma signatures. Inflammation signatures in combination with healthy B cell and tonsil characteristics associate with better overall survival rates, while proliferation in combination with inflammation and plasma cell characteristics worsens it. A phenotypic similarity tree is presented that reveals possible progression paths along the transcriptional dimensions. Our analysis provided a novel look on the transition range between FL and DLBCL, on DLBCL with poor prognosis showing expression patterns resembling that of Burkitt's lymphoma and particularly on 'double-hit' MYC and BCL2 transformed lymphomas., Conclusions: The transcriptome map provides a tool that aggregates, refines and visualizes the data collected in the MMML study and interprets them in the light of previous knowledge to provide orientation and support in current and future studies on lymphomas and on other cancer entities.

Normal and malignant B cells carry rearranged immunoglobulin (Ig) variable region genes, which due to their practically limitless diversity represent ideal clonal markers for these cells. We describe here an approach to isolate single cells from frozen tissue sections by microdissection using a laser-based method. From the isolated cells, rearranged IgH and Igκ genes are amplified in a semi-nested PCR approach, using a collection of V gene subgroup-specific primers recognizing nearly all V genes together with primers for the J genes. By sequence analysis of V region genes from distinct cells, the clonal relationship of the B lineage cells can unequivocally be determined and related to the histological distribution of the cells. The approach is also useful to determine V, D, and J gene usage. Moreover, the presence and pattern of somatic Ig V gene mutations give valuable insight into the stage of differentiation of the B cells.

Angioimmunoblastic T-cell lymphomas (AITLs) frequently carry mutations in the TET2 and IDH2 genes. TET2 mutations represent early genetic lesions as they had already been detected in haematopoietic precursor cells of AITL patients. We show by analysis of whole-tissue sections and microdissected PD1 + cells that the frequency of TET2-mutated AITL is presumably even higher than reported (12/13 cases in our collection; 92%). In two-thirds of informative AITLs (6/9), a fraction of B cells was also TET2-mutated. Investigation of four AITLs by TET2 and IGHV gene sequencing of single microdissected B cells showed that between 10% and 60% of polyclonal B cells in AITL lymph nodes harboured the identical TET2 mutations of the respective T-cell lymphoma clone. Thus, TET2-mutated haematopoietic precursor cells in AITL patients not only give rise to the T-cell lymphoma but also generate a large population of mutated mature B cells. Future studies will show whether this is a reason why AITL patients frequently also develop B-cell lymphomas. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) presents different histopathologic growth patterns, including atypical forms showing overlapping histopathologic and clinical features with T-cell/histiocyte-rich large B-cell lymphoma (THRLBCL). Because growth patterns are associated with vessel distribution, the aim of the present study was to compare angiogenesis in different NLPHL patterns with THRLBCL as well as other lymphomas. Atypical variants of NLPHL and THRLBCL (n=10 per group) both showed a low microvessel density (MVD; 1.16-1.31/μm 2 ) with a diffuse vessel distribution. In contrast, in typical NLPHL (n=10), follicular areas with low MVD were retained, whereas an increase in vessels in the interfollicular areas was observed (MVD 1.35/μm 2 ). THRLBCL and typical NLPHL could additionally be distinguished by differences in their molecular angiogenesis signature. Furthermore, the number of intravascular T cells was significantly reduced in THRLBCL (0.0028 T cells/mm 2 vessel area) when compared with typical NLPHL (0.0059 T cells/mm 2 vessel area), potentially reflecting the different composition of the microenvironment in these 2 lymphoma entities. The results of our study reveal a similar vascular pattern and angiogenesis behavior in atypical NLPHL variants and THRLBCL in contrast to the retained follicular pattern in typical NLPHL., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We recently reported a truncating deletion in the NFKBIE gene, which encodes IκBε, a negative feedback regulator of NF-κB, in clinically aggressive chronic lymphocytic leukemia (CLL). Because preliminary data indicate enrichment of NFKBIE aberrations in other lymphoid malignancies, we screened a large patient cohort (n = 1460) diagnosed with different lymphoid neoplasms. While NFKBIE deletions were infrequent in follicular lymphoma, splenic marginal zone lymphoma, and T-cell acute lymphoblastic leukemia (<2%), slightly higher frequencies were seen in diffuse large B-cell lymphoma, mantle cell lymphoma, and primary central nervous system lymphoma (3% to 4%). In contrast, a remarkably high frequency of NFKBIE aberrations (46/203 cases [22.7%]) was observed in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (3/11 cases [27.3%]). NFKBIE-deleted PMBL patients were more often therapy refractory (P = .022) and displayed inferior outcome compared with wild-type patients (5-year survival, 59% vs 78%; P = .034); however, they appeared to benefit from radiotherapy (P =022) and rituximab-containing regimens (P = .074). NFKBIE aberrations remained an independent factor in multivariate analysis (P = .003) and when restricting the analysis to immunochemotherapy-treated patients (P = .008). Whole-exome sequencing and gene expression profiling verified the importance of NF-κB deregulation in PMBL. In summary, we identify NFKBIE aberrations as a common genetic event across B-cell malignancies and highlight NFKBIE deletions as a novel poor-prognostic marker in PMBL., (© 2016 by The American Society of Hematology.)

In rare instances, two distinct lymphomas concurrently occur in a patient. Such composite lymphomas can be combinations of two non-Hodgkin lymphomas or a combination of a non-Hodgkin lymphoma and a Hodgkin's lymphoma. Composite lymphomas pose a particular diagnostic challenge, and there are currently no agreed standards for treatment. Combined B-cell non-Hodgkin lymphomas are often clonally unrelated. However, in many composite non-Hodgkin lymphomas and Hodgkin's lymphomas, the tumours are clonally related. In most of these instances, the malignant clones developed separately from a common precursor, usually a germinal centre B cell. This finding suggests a scenario in which the common premalignant precursor had acquired shared transforming events, and the two distinct lymphomas developed from descendants of that precursor after acquiring additional separate transforming events. Findings from molecular studies support this notion. Hence, clonally related composite lymphomas are elegant models to study the multistep transformation process in lymphomagenesis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

There is ample epidemiologic evidence for an association of chronic hepatitis C virus (HCV) infection with B-cell non-Hodgkin lymphoma (B-NHL). B-NHL subtypes most frequently associated with HCV are marginal zone lymphoma and diffuse large B-cell lymphoma. The most convincing evidence for a causal relationship between HCV infection and lymphoma development is the observation of B-NHL regression after HCV eradication by antiviral therapy (AVT). In fact, for indolent HCV-associated B-NHL, first-line AVT instead of standard immune-chemotherapy might be considered. Molecular mechanisms of HCV-NHL development are still poorly understood. Three general theories have emerged to understand the HCV-induced lymphomagenesis: (1) continuous external stimulation of lymphocyte receptors by viral antigens and consecutive proliferation; (2) HCV replication in B cells with oncogenic effect mediated by intracellular viral proteins; (3) permanent B-cell damage, e.g., mutation of tumor suppressor genes, caused by a transiently intracellular virus ("hit and run" theory). This review systematically summarizes the data on epidemiology, interventional studies, and molecular mechanisms of HCV-associated B-NHL., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell-specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non-B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

Proliferation and survival of Hodgkin and Reed/Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (cHL), are dependent on constitutive activation of nuclear factor kappaB (NF-kappaB). NF-kappaB activation through various stimuli is negatively regulated by the zinc finger protein A20. To determine whether A20 contributes to the pathogenesis of cHL, we sequenced TNFAIP3, encoding A20, in HL cell lines and laser-microdissected HRS cells from cHL biopsies. We detected somatic mutations in 16 out of 36 cHLs (44%), including missense mutations in 2 out of 16 Epstein-Barr virus-positive (EBV(+)) cHLs and a missense mutation, nonsense mutations, and frameshift-causing insertions or deletions in 14 out of 20 EBV(-) cHLs. In most mutated cases, both TNFAIP3 alleles were inactivated, including frequent chromosomal deletions of TNFAIP3. Reconstitution of wild-type TNFAIP3 in A20-deficient cHL cell lines revealed a significant decrease in transcripts of selected NF-kappaB target genes and caused cytotoxicity. Extending the mutation analysis to primary mediastinal B cell lymphoma (PMBL), another lymphoma with constitutive NF-kappaB activity, revealed destructive mutations in 5 out of 14 PMBLs (36%). This report identifies TNFAIP3 (A20), a key regulator of NF-kappaB activity, as a novel tumor suppressor gene in cHL and PMBL. The significantly higher frequency of TNFAIP3 mutations in EBV(-) than EBV(+) cHL suggests complementing functions of TNFAIP3 inactivation and EBV infection in cHL pathogenesis.

Lymphomas are assumed to originate at different stages of lymphocyte development through chromosomal aberrations. Thus, different lymphomas resemble lymphocytes at distinct differentiation stages and show characteristic morphologic, genetic, and transcriptional features. Here, we have performed a microarray-based DNA methylation profiling of 83 mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) characterized for their morphologic, genetic, and transcriptional features, including molecular Burkitt lymphomas and diffuse large B-cell lymphomas. Hierarchic clustering indicated that methylation patterns in maB-NHLs were not strictly associated with morphologic, genetic, or transcriptional features. By supervised analyses, we identified 56 genes de novo methylated in all lymphoma subtypes studied and 22 methylated in a lymphoma subtype-specific manner. Remarkably, the group of genes de novo methylated in all lymphoma subtypes was significantly enriched for polycomb targets in embryonic stem cells. De novo methylated genes in all maB-NHLs studied were expressed at low levels in lymphomas and normal hematopoietic tissues but not in nonhematopoietic tissues. These findings, especially the enrichment for polycomb targets in stem cells, indicate that maB-NHLs with different morphologic, genetic, and transcriptional background share a similar stem cell-like epigenetic pattern. This suggests that maB-NHLs originate from cells with stem cell features or that stemness was acquired during lymphomagenesis by epigenetic remodeling.

The spectrum of entities, the therapeutic strategy, and the outcome of mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHLs have been studied in detail, data on molecular profiling of pediatric maB-NHLs are hitherto lacking. We analyzed 65 cases of maB-NHL from patients up to 18 years of age by gene expression profiling, matrix comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and immunohistochemistry. The majority of the analyzed pediatric patients were treated within prospective trials (n = 49). We compared this group to a series of 182 previously published cases of adult maB-NHL. Gene expression profiling reclassified 31% of morphologically defined diffuse large B-cell lymphomas as molecular Burkitt lymphoma (mBL). The subgroups obtained by molecular reclassification did not show any difference in outcome in children treated with the NHL-Berlin-Frankfurt-Muenster (BFM) protocols. No differences were detectable between pediatric and adult mBL with regard to gene expression or chromosomal imbalances. This is the first report on molecular profiling of pediatric B-NHL showing mBL to be much more prominent in children than suggested by morphologic assessment. Based on molecular profiling mBL is a molecularly homogeneous disease across children and adults.

Secondary lymphomas occurring in the setting of angioimmunoblastic T-cell lymphoma (AILT) are considered to be rare. Their occurrence has been attributed to Epstein-Barr virus (EBV)-associated lymphoproliferations. A previous study detected a dysregulated hypermutation process in B-cells of AILT. The present study aimed at estimating the frequency of B-cell lymphomas in AILT. By studying the expression of EBV and activation-induced cytidine deaminase (AID) as an indicator of hypermutating cells, we assessed whether B-cell lymphoproliferations in AILT were strictly associated with EBV and whether hypermutation might contribute to lymphomagenesis. Among 161 cases of AILT, diagnosed between 1996 and 2005 at the lymph node registry, Frankfurt, Germany, 19 cases were detected that also had B-cell non-Hodgkin lymphoma (NHL) and two cases had classical Hodgkin lymphoma (HL). EBV was detected in tumour cells of 7/18 NHL and both HL, suggesting that factors other than EBV contribute to lymphomagenesis. AID was expressed in AILT in large cells disseminated in the tissue, implying that the process of somatic hypermutation is ongoing in AILT, although the GC architecture is disrupted. This might be relevant in the development of secondary lymphomas.

Mediastinal large B-cell (MBL) and classical Hodgkin lymphoma (HL) have several pathogenic mechanisms in common. As we recently observed aberrant tyrosine kinase (TK) activities in HL, we now analysed also MBL for such activities. Indeed, MBL and HL were the only B-cell lymphomas where elevated cellular phospho-tyrosine contents were typical features. Three TKs, JAK2, RON and TIE1, not expressed in normal B cells, were each expressed in about 30% of MBL cases, and 75% of cases expressed at least one of the TKs. Among the intracellular pathways frequently triggered by TKs, the PI3K/AKT pathway was activated in about 40% of MBLs and essential for survival of MBL cell lines, whereas the RAF/mitogen-activated protein kinase pathway seemed to be inhibited. No activating mutations were detected in the three TKs in MBL cell lines and primary cases. RON and TIE1 were each also expressed in about 35% and JAK2 in about 53% of HL cases. JAK2 genomic gains are frequent in MBL and HL but we observed no strict correlation of JAK2 genomic status with JAK2 protein expression. In conclusion, aberrant TK activities are a further shared pathogenic mechanism of MBL and HL and may be interesting targets for therapeutic intervention.

Members of the BCL-2 gene family are well known for their role in the pathogenesis of B-cell lymphomas in humans and in mouse models. A recent report that knockout mice deficient for the proapoptotic BCL-2 family member gene BAD frequently develop B-cell lymphomas prompted us to analyze a large collection of human B-cell lymphomas for inactivating mutations in the BAD gene. All 3 exons of the BAD gene were amplified and directly sequenced. The 81 lymphomas analyzed included 16 cases of B-cell chronic lymphocytic leukemia, 11 mantle-cell lymphomas, 10 follicular lymphomas, 7 MALT lymphomas, 8 Burkitt's lymphoma cell lines, 3 cell lines of multiple myeloma, 15 cases and 4 cell lines of diffuse large B-cell lymphoma and 7 Hodgkin's lymphoma lines. No mutations were found in any of the cases. We conclude that mutations in the BAD gene do not play a role in the pathogenesis of the major subtypes of human B-cell lymphomas., (Copyright 2006 Wiley-Liss, Inc.)

Rearrangements of the MALT1 gene by the t(11;18)(q21;q21) and t(14;18)(q32;q21) are the most frequent structural chromosomal abnormalities in MALT lymphomas. These translocations lead to fusions of BIRC3-MALT1 and IGH-MALT1 respectively, and activate the NF-kappaB pathway. Among 122 diffuse large B-cell lymphomas and 28 Burkitt's lymphomas screened by interphase FISH, we found two cases with a break within MALT1, but without a t(11;18) or a t(14;18). Molecular genetic analyses in one of these cases revealed a novel "in frame" fusion of exon 9 of MALT1 and exon 9 of the microtubule-associated protein 4 (MAP4) gene. The translocation was accompanied by a deletion of MALT1 sequences distal to the breakpoint including the caspase-like domain, which is essential for activation of NF-kappaB. As a result of the deletion, the reciprocal 5'MAP4-3'MALT1 transcript was not present, demonstrating that the 5'MALT1-3'MAP4 fusion represents the pathogenetically relevant transcript. Immunohistochemistry with amino-terminal and carboxy-terminal MALT1 antibodies, indicated a strong expression of the chimeric MALT1-MAP4 protein. Moreover, NF-kappaB activation was not increased in this case as shown by the levels of IkappaBalpha phosphorylation and NEMO ubiquitination. Our data demonstrate that the pathogenetic consequences of the novel MALT1-MAP4 fusion are different from those of the known MALT1-associated chromosomal rearrangements and do not involve NF-kappaB activation., ((c) 2006 Wiley-Liss, Inc.)

SLP65 represents a critical component in (pre-) B cell receptor signal transduction but is compromised in a subset of pre-B cell-derived acute lymphoblastic leukemia. Based on these findings, we investigated (i.) whether SLP65-deficiency also occurs in mature B cell-derived lymphoma and (ii.) whether SLP65-deficient B cell lymphoma cells use an alternative B cell receptor signaling pathway in the absence of SLP65. Indeed, expression of SLP65 protein was also missing in a fraction of B cell lymphoma cases. While SLP65 is essential for B cell receptor-induced Ca2+ mobilization in normal B cells, B cell receptor engagement in SLP65-deficient as compared to SLP65-reconstituted B cell lymphoma cells resulted in an accelerated yet shortlived Ca2+-signal. B cell receptor engagement of SLP65-deficient lymphoma cells involves SRC kinase activation, which is critical for B cell receptor-dependent Ca2+-mobilisation in the absence but not in the presence of SLP65. As shown by RNA interference, the SRC kinase LYN is required for B cell receptor-induced Ca2+ release in SLP65-deficient B cell lymphoma cells but dispensable after SLP65-reconstitution. B cell receptor engagement in SLP65-deficient B cell lymphoma cells also resulted in tyrosine-phosphorylation of the proliferation- and survival-related MAPK1 and STAT5 molecules, which was sensitive to silencing of the SRC kinase LYN. Inhibition of SRC kinase activity resulted in growth arrest and cell death specifically in SLP65-deficient lymphoma cells. These findings indicate that LYN can short-circuit conventional B cell receptor signaling in SLP65-deficient B cell lymphoma cells and thereby promote activation of survival and proliferation-related molecules.

Jaw1, also known as lymphoid-restricted membrane protein (LRMP), is an endoplasmic reticulum-associated protein. High levels of Jaw1/LRMP mRNA have been found in germinal centre B-cells and in diffuse large B-cell lymphomas of 'germinal centre' subtype. This paper documents Jaw1/LRMP expression at the protein level in human tissues by immunohistochemical and western blotting analysis using an antibody reactive with paraffin-embedded tissues. Jaw1/LRMP was highly expressed in germinal centre B-cells (in keeping with gene expression data), in 'monocytoid B-cells', and in splenic marginal zone B-cells. It was absent, or present at only low levels, in mature T-cells, although cortical thymocytes were weakly positive. Among lymphoid neoplasms, Jaw1/LRMP was found in germinal centre-derived lymphomas (follicle centre lymphoma, Burkitt's lymphoma, lymphocyte-predominant Hodgkin's disease) but not in T-cell neoplasms (with the exception of a single T lymphoblastic lymphoma). Classical Hodgkin's disease and myeloma lacked Jaw1/LRMP but many cases of chronic lymphocytic leukaemia (but not mantle zone lymphoma) were Jaw1/LRMP-positive. Approximately half of the marginal zone lymphomas were Jaw1/LRMP-positive. In diffuse large B-cell lymphomas, Jaw1/LRMP was found in three-quarters (24/32) of the cases classified phenotypically as being of 'germinal centre' type, but it was also expressed in almost half (13/28) of the 'non-germinal centre' cases. A similar proportion of 'non-germinal centre' cases were positive for the protein products of two other genes expressed highly in germinal centre cells (HGAL/GCET2 and PAG). The fact that all three of these proteins are expressed in a significant proportion of diffuse large B-cell lymphomas assigned to the 'non-germinal centre' category indicates that the immunophenotypic categorization of diffuse large B-cell lymphoma according to cellular origin may be more complicated than currently understood. Finally, the expression of Jaw1/LRMP in other types of lymphoma and in non-lymphoid tissues/tumours may be of interest in differential diagnosis and research., (Copyright 2006 Pathological Society of Great Britain and Ireland.)

Background: The distinction between Burkitt's lymphoma and diffuse large-B-cell lymphoma is unclear. We used transcriptional and genomic profiling to define Burkitt's lymphoma more precisely and to distinguish subgroups in other types of mature aggressive B-cell lymphomas., Methods: We performed gene-expression profiling using Affymetrix U133A GeneChips with RNA from 220 mature aggressive B-cell lymphomas, including a core group of 8 Burkitt's lymphomas that met all World Health Organization (WHO) criteria. A molecular signature for Burkitt's lymphoma was generated, and chromosomal abnormalities were detected with interphase fluorescence in situ hybridization and array-based comparative genomic hybridization., Results: We used the molecular signature for Burkitt's lymphoma to identify 44 cases: 11 had the morphologic features of diffuse large-B-cell lymphomas, 4 were unclassifiable mature aggressive B-cell lymphomas, and 29 had a classic or atypical Burkitt's morphologic appearance. Also, five did not have a detectable IG-myc Burkitt's translocation, whereas the others contained an IG-myc fusion, mostly in simple karyotypes. Of the 176 lymphomas without the molecular signature for Burkitt's lymphoma, 155 were diffuse large-B-cell lymphomas. Of these 155 cases, 21 percent had a chromosomal breakpoint at the myc locus associated with complex chromosomal changes and an unfavorable clinical course., Conclusions: Our molecular definition of Burkitt's lymphoma clarifies and extends the spectrum of the WHO criteria for Burkitt's lymphoma. In mature aggressive B-cell lymphomas without a gene signature for Burkitt's lymphoma, chromosomal breakpoints at the myc locus were associated with an adverse clinical outcome., (Copyright 2006 Massachusetts Medical Society.)

Aims: To investigate whether an antibody against an intracellular epitope can detect CD19 in routine biopsy specimens and thus to document in detail its expression in human lymphomas., Method and Results: A polyclonal antibody to the C terminus of CD19 was used to immunostain paraffin-embedded samples of normal and neoplastic lymphoid tissues. CD19 was widely expressed in normal B cells and in extramedullary plasma cells. It was found in most B-cell neoplasms, but expression in follicular lymphoma was weak (33/69) or negative (four cases). Similarly, CD19 expression in diffuse large B-cell lymphomas was weak (28/56) or negative (eight cases). In T-cell-rich B-cell lymphomas, CD19 was also weak (4/10) or negative (three cases). CD19 was often absent in post-transplant B lymphoproliferative disease, classical Hodgkin's disease and plasma cell neoplasms. An unexpected finding was the frequent absence of CD19 in the neoplastic cells in lymphocyte predominant Hodgkin's disease., Conclusions: CD19 can now be detected in routine biopsy specimens. In contrast to the classical pan-B marker CD20, CD19 is not always strongly expressed in B-cell neoplasms. Furthermore, the lymphocytic and histiocytic (L&H) cells of lymphocyte predominant Hodgkin's disease (which express most B-cell-associated markers) commonly lack CD19.

Two microarray studies of mediastinal B cell lymphoma have shown that this disease has a distinct gene expression profile, and also that this is closest to the pattern seen in classical Hodgkin's disease. We reported previously an immunohistologic study in which the loss of intracellular B cell-associated signaling molecules in Reed-Sternberg cells was demonstrated, and in this study we have investigated the expression of the same components in more than 60 mediastinal B cell lymphomas. We report that these signaling molecules are frequently present, and in particular that Syk, BLNK and PLC-gamma2 (absent from Reed-Sternberg cells) are present in the majority of mediastinal B cell lymphomas. The overall pattern of B cell signaling molecules in this disease is therefore closer to that of diffuse large B cell lymphoma than to Hodgkin's disease, and is consistent with a common cell of origin as an explanation of the similar gene expression profiles.

FCRL (also known as FREB and FcRX) is a recently described member of the family of Fc receptors for immunoglobulin G (IgG). In the present study we analysed its expression in normal and neoplastic lymphoid tissue using immunohistochemical techniques. FCRL was preferentially expressed in a proportion of germinal centre cells and, more weakly, in mantle zone B cells. In addition, strong labelling was observed in marginal zone B cells in the spleen, representing one of the few markers for this cell type. The majority of cases of small B-cell lymphoma, diffuse large B-cell lymphoma and lymphocyte predominance Hodgkin's disease were positive for FCRL. However, the number of positive cells varied widely, and in consequence we could not define a cut-off that distinguished subsets of diffuse large B-cell lymphoma. Our results also showed that FCRL tended to be negative in T-cell-rich B-cell lymphoma and in classical Hodgkin's disease. FCRL may therefore represent a novel marker for normal B cells (e.g. splenic marginal zone cells) and may also be useful as a potential marker of B-cell neoplasms.

Composite lymphomas are rare combinations of Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma in the same patient, where clonal relatedness has been observed in most of the few cases analyzed. Here, we report a composite classical HL and diffuse large B-cell lymphoma (DLBCL) with interesting molecular features. Micromanipulation of single cells and analysis of V gene rearrangements revealed clonal relatedness with shared and distinct mutations, indicative of derivation from a common germinal center (GC) B-cell precursor and also of further development of both lymphomas in a GC. In the DLBCL, a very high mutation load, including inactivating mutations, and two copies of the same clonal rearrangement with different mutations in single cells were observed. Intriguingly, in the DLBCL precursor somatic hypermutation activity continued after acquisition of destructive V gene mutations, a feature previously found only in Epstein-Barr virus (EBV) infected B-cell expansions. Furthermore, we found evidence of light-chain receptor revision in the lymphoma precursor during a GC reaction. Re-expression of the V(D)J recombination machinery may enhance genomic instability in GC B cells and contribute to lymphomagenesis.

Background: Hodgkin's disease and non-Hodgkin's B-cell lymphoma occasionally occur in the same patient. The identification of a common precursor of the two types of lymphoma would show definitively that Reed-Sternberg cells originate from B cells., Methods: We studied lymphomas from two patients, one with a composite lymphoma (classic Hodgkin's disease and a follicular lymphoma in the same lymph node) and the other with a T-cell-rich B-cell lymphoma that was followed by classic Hodgkin's disease. Single Reed-Sternberg cells and non-Hodgkin's lymphoma cells from frozen sections were micromanipulated. The rearranged immunoglobulin variable-region genes (V genes) of the heavy and light chains were amplified by the polymerase chain reaction from genomic DNA and sequenced., Results: In both patients, the Reed-Sternberg cells were related clonally to the non-Hodgkin's lymphoma B cells. The V genes carried somatic mutations (a hallmark of germinal-center B cells and their descendants). In both patients, some somatic mutations were shared by the Reed-Sternberg and non-Hodgkin's lymphoma cells, whereas other somatic mutations were found exclusively in one or the other cell type., Conclusions: In two patients with classic Hodgkin's disease and non-Hodgkin's B-cell lymphoma, we identified a common B-cell precursor, probably a germinal-center B-cell, for both lymphomas. This finding suggests that the two types of lymphoma underwent both shared and distinct transforming events and provides proof of the B-cell derivation of Reed-Sternberg cells in classic Hodgkin's disease.

T-cell-rich B-cell lymphoma (TCRBCL) belongs to the group of diffuse large cell lymphomas (DLL). It is characterized by a small number of tumor B cells among a major population of nonmalignant polyclonal T cells. To identify the developmental stage of the tumor progenitor cells, we micromanipulated the putative neoplastic large CD20(+) cells from TCRBCLs and amplified and sequenced immunoglobulin (Ig) V gene rearrangements from individual cells. In six cases, clonal Ig heavy, as well as light chain, gene rearrangements were amplified from the isolated B cells. All six cases harbored somatically mutated V gene rearrangements with an average mutation frequency of 15.5% for heavy (VH) and 5.9% for light (VL) chains and intraclonal diversity based on somatic mutation. These findings identify germinal center (GC) B cells as the precursors of the transformed B cells in TCRBCL. The study also exemplifies various means how Ig gene rearrangements can be modified by GC B cells or their malignant counterparts in TCRBCL: In one case, the tumor precursor may have switched from kappa to lambda light chain expression after acquiring a crippling mutation within the initially functional kappa light chain gene. In another case, the tumor cells harbor two in-frame VH gene rearrangements, one of which was rendered nonfunctional by somatic mutation. Either the tumor cell precursor entered the GC with two potentially functional in-frame rearrangements or the second VHDHJH rearrangement occurred in the GC after the initial in-frame rearrangement was inactivated by somatic mutation. Finally, in each of the six cases, at least one cell contained two (or more) copies of a clonal Ig gene rearrangement with sequence variations between these copies. The presence of sequence variants for V region genes within single B cells has so far not been observed in any other normal or transformed B lymphocyte. Fluorescence in situ hybridization (FISH) points to a generalized polyploidy of the tumor cells.

Five cases of monocytoid B cell lymphoma (MBCL) were analyzed for somatic mutations in the rearranged V region genes. Somatic mutations were found in four of the five cases, whereas one unusual CD5+ lymphoma harbored unmutated V region genes. Since somatic mutations are introduced into V region genes of antigen-activated B cells in the course of T cell-dependent immune responses, these results suggest a derivation of the tumor B cells in MBCL from antigen-experienced mature B cells. An analysis of the kappa-deleting element in two of the cases in which mutated VH but unmutated and nonfunctional V kappa gene rearrangements were found suggests that somatic hypermutation does not take place in human rearranged V kappa region genes when the C kappa gene and the kappa enhancers have been deleted in cis by rearrangement of the kappa-deleting element.

B-cell non-Hodgkin's lymphomas with a marked preponderance of reactive T cells, so-called T-cell rich B-cell lymphomas (TCRBCLs), can be morphologically confused with Hodgkin's disease (HD). To establish helpful distinguishing features in paraffin sections, 10 cases of L26-positive, CD15-negative HD and 10 cases of TCRBCL were compared; 4 cases of HD had morphologic features of the nodular lymphocyte predominant (LP) type. Nine of 10 cases of HD contained fewer than 20 mitoses/20 high power fields (hpf) and only 1 had pericapsular involvement. In contrast, 9 of 10 TCRBCL had greater than 20 mitoses/20 hpf and 7 had perinodal infiltration. HDLP was easily distinguished from TCRBCL by the expanded dendritic meshworks outlining the L & H nodules and the high content of CD57-positive lymphocytes. The remaining 6 cases of non-LP L26-positive HD had a relatively distinctive immunostaining pattern, with absence of CD45 and discordant reactivity for L26 and Ki-B5 in Reed-Sternberg cells and variants. Only 3 cases of TCRBCL had a similar CD45 and L26/Ki-B5 immunostaining pattern, and these could be distinguished by demonstrable cytoplasmic light-chain restriction. These results show that evaluation of the mitotic count, pericapsular involvement, and immunohistochemical staining patterns for Ki-M4p, CD57, L26/Ki-B5, and CD45 can help to discriminate TCRBCL from L26-positive HD when only fixed material is available.

To distinguish reactive versus neoplastic monocytoid B-cell (MBC) proliferations, the clonality of MBC was examined in paraffin-embedded tissues by in situ hybridization (ISH) of immunoglobulin (Ig) light chain messenger RNA (mRNA) with sensitive oligonucleotide probes in 26 cases. They included 13 cases of lymphadenitis with MBC reaction and 13 cases of nodal (n = 8) and extranodal (n = 5) monocytoid B-cell lymphoma (MBCL). Two cases represented a composite lymphoma showing a centroblastic-centrocytic and MBCL component. The clonality of MBC infiltrates could be demonstrated in 16 of 26 (61.5%) cases by immunostaining for Ig light chains and in all (100%) cases by ISH. Neoplastic MBC usually expressed a faint-to-moderate light chain restriction of mRNA, whereas some MBC (10% to 30% of total MBCL population) showed a strong positivity irrespective of plasmacytoid differentiation as indicated by Ig immunostaining (present in 9 of 13 cases). Reactive MBC expressed a faint kappa and lambda light-chain mRNA positivity. Five percent to 20% of total reactive MBC showed also a strong positivity for both Ig light chain mRNA, although only a minor part of these cells (7 of 13 cases) expressed polyclonal Ig by immunohistochemistry. These results indicate that (1) both reactive and neoplastic MBC can differentiate into plasma cells; and (2) a relatively high percentage of reactive and neoplastic MBC show a detectable mRNA transcription, but not a corresponding Ig synthesis. Either the Ig detection is not sensitive enough or these cells might be in an early differentiation phase, where the Ig production has not yet started.

To determine which morphologic criteria are most useful in distinguishing reactive from malignant monocytoid B cells (MBCs), we compared 16 monoclonal cases (11 nodal, five extranodal) of monocytoid B-cell lymphoma (MBCL) with 12 cases of various reactive diseases in which MBCs were polyclonal. The results of our study showed that in MBCL the MBC component was the predominant architectural finding and that there was confluence of MBCs in all but one case. In contrast, the MBC component did not predominate in the reactive group (P less than .000001) and focal confluence was seen in only one case. A cytologic comparison showed that in MBCL areas there were more large transformed (prominent nucleolated) MBCs (P = .003), a higher mitotic rate (P = .03), and more nuclear irregularities (P = .007) than were present in the reactive group. In addition, evolution to an aggressive histologic type was found in four cases of MBCL. Our results also revealed concomitant multiple, monoclonal, morphologically distinct populations in other compartments (follicular center cells in seven, mantle cells in five, small lymphocytes in five, and plasma cells in 11). These unique findings can be reconciled by postulating (1) that the simultaneous presence of these diverse cytologic types represents morphologic expressions of a B cell whose population is in different phases of its cell cycle and/or its evolution or (2) that the histogenesis of MBCL is possibly from a nodal pluripotent B-stem cell that can differentiate directly into these various cytologic types.

We have investigated seven cases of large cell lymphomas (LCL) developing simultaneously or metachronously to nodular lymphocyte-predominant Hodgkin's disease (nodular paragranuloma, NP) for the presence of EBV and the chromosomal translocation t(14;18) by use of the polymerase chain reaction (PCR). The expression of the bcl-2 oncogene product in these cases and in five cases of progressive transformation of germinal centres as a potential precursor of NP was detected immunohistochemically with the monoclonal antibodies bcl-2-100 and bcl-2-124. All cases investigated were negative for EBV genomic material. The chromosomal translocation t(14;18) was also absent. Expression of the bcl-2 oncogene could be detected only in one case of nodular paragranuloma and in an unrelated case of LCL. Hence, LCL developing out of NP differ from other germinal center derived high-grade lymphomas.

Resection specimens from 145 patients with primary B-cell gastric lymphoma at stage IE (n = 88) and at stage IIE (n = 57) were investigated. Histologically, low-grade malignant B-cell lymphomas arising from the mucosa-associated lymphoid tissue, including immunocytoma (n = 71), could be distinguished from high-grade malignant B-cell lymphomas with (n = 25) and without (n = 49) evidence of a low-grade component. The very rare low-grade B-cell lymphomas of centroblastic-centrocytic, centrocytic, and plasmacytic type were not considered. All patients had undergone primary gastric resection, and 65 received additional chemotherapy (n = 33), radiotherapy (n = 22), or both (n = 10). Actuarial overall survival rates calculated by the Kaplan-Meier life-table method were 76% after 5 years and 58% after 10 years. According to the Mantel test and a multivariate analysis using the Cox regression method, patients at stage IE had a significantly better survival probability than those at stage IIE (P less than 0.0001); 5-year survival rates were 87% and 61%, respectively. The survival probability for low-grade malignant lymphomas was significantly better than for tumors with secondary high-grade transformation (P less than 0.05) or for primary high-grade lymphomas (P less than 0.0001), whereas the two high-grade groups were not significantly different. Five-year survival rates were 91% for low-grade, 73% for secondary high-grade, and 56% for primary high-grade malignant lymphomas. Retrospectively, no significantly different survival rates were found between patients who had undergone gastric resection alone and patients who had received additional treatment. However, survival analyses showed that classification and grading according to the histopathological concept of mucosa-associated lymphoid tissue-derived gastric lymphomas into low-grade B-cell lymphomas of mucosa-associated lymphoid tissue type and high-grade B-cell lymphomas with or without evidence of a low-grade component has great prognostic relevance.

In the search for immunoreagents appropriate for the histopathologic diagnosis of malignant B-cell lymphomas in routinely processed paraffin sections, a new monoclonal antibody, Ki-B5, was generated using a high-grade B-cell lymphoma as the immunogene. Ki-B5 is a mouse IgG1/kappa that recognizes five protein fractions of about 84, 82, 55, 48, and 27 Kd after biosynthetic radiolabeling and immunoprecipitation. Protein fractions with the molecular weights of approximately 84 and 82 Kd were expressed on the cell surface and show that Ki-B5 is probably unrelated to CD45. It was possible through electron microscopy to visualize the membrane-bound portion of Ki-B5. Extensive immunohistologic studies on normal human tissue and various neoplasias demonstrated the high specificity of Ki-B5 to normal human B cells and a minor subgroup of plasma cells. Except for ML-2, which is a myelomonocytic human cell line, Ki-B5 exclusively recognized the B-cell lineage, including EB-3, BALL-1, and NALM-1. All carcinomas, sarcomas, and malignant melanomas tested with Ki-B5 were negative. Although normal granulocytes and monocytes were constantly negative, three of eight myelomonocytic leukemias coreacted with this antibody. Eight of the 57 T-cell lymphomas studied were positive to Ki-B5. Five were classified as lymphoblastic, two represented T8-CLL, and one was classified as immunoblastic T-cell lymphoma. Only 3 of 126 cases of B-cell lymphoma, including rare types not considered in the current classifications, were negative to Ki-B5. Plasmacytomas were also negative, except for one case. Irrespective of the cases of lymphoblastic lymphoma and plasmacytoma, Ki-B5 represents a new monoclonal antibody appropriate for the diagnosis and immunophenotyping of malignant lymphomas in routinely processed paraffin sections.

This study surveyed 97 cases of low grade B-cell lymphoma (LGBL) of the gastrointestinal tract (GIT) by conventional morphology and immunohistochemistry, focusing on the most frequent subtype: the so-called LGBL of the mucosa-associated lymphoid tissue (MALT). Special reference was made to the follicular dendritic cells (FDCs) selectively visualized by a new paraffin-resistant monoclonal antibody Ki-FDC1p. LGBL of the MALT accounted for 83 cases. The diagnosis was based on (a) a characteristic cytology, which included centrocytoid cells, a varying degree of plasma cell differentiation, and some blasts; (b) the presence of lymphoepithelial lesions; and (c) the occurrence of two types of follicles easily detectable with Ki-FDC1p. Some were restricted to the mucosa, contained normal germinal center cells, and were indistinguishable from reactive follicles. Others consisted of small clusters of FDCs, were randomly distributed throughout the tumor, and escaped detection in conventional stainings. Such small clusters of FDCs were found to be restricted to LGBL of the MALT, not occurring in other types of LGBL, and were interpreted as tumor-associated abortive follicles discernable from residues of reactive follicles due to their cellular constituents, localization, and distribution pattern. Eight cases showed closed sheets of blasts and were classified as high grade malignant lymphoma secondary to LGBL of the MALT. In two cases the LGBL of the MALT were restricted to the mucosa, in 31 cases the submucosa was also infiltrated, and in the remaining 50 cases the infiltration also involved deeper wall layers of the GIT.(ABSTRACT TRUNCATED AT 250 WORDS)