Post-germinal Center B-cells Research Articles

AB0027 B-Cell Activating Factor (BAFF) Binding Receptors (BBR) on B Cells: Characterization in Patients with Rheumatoid Arthritis (RA) Receiving Biological Therapies: Anti-TNF, Anti-Il6r and Anti-Ctla4: A Longitudinal Study

BackgroundThe treatment of rheumatoid arthritis (RA) has improved in the last years because of both: an increased understanding of its pathogenesis and the use of biological therapies. It is well...

SPIB and BATF provide alternate determinants of IRF4 occupancy in diffuse large B-cell lymphoma linked to disease heterogeneity.

Interferon regulatory factor 4 (IRF4) is central to the transcriptional network of activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), an aggressive lymphoma subgroup defined by gene expression profiling. Since cofactor association modifies transcriptional regulatory input by IRF4, we assessed genome occupancy by IRF4 and endogenous cofactors in ABC-DLBCL cell lines. IRF4 partners with SPIB, PU.1 and BATF genome-wide, but SPIB provides the dominant IRF4 partner in this context. Upon SPIB knockdown IRF4 occupancy is depleted and neither PU.1 nor BATF acutely compensates. Integration with ENCODE data from lymphoblastoid cell line GM12878, demonstrates that IRF4 adopts either SPIB- or BATF-centric genome-wide distributions in related states of post-germinal centre B-cell transformation. In primary DLBCL high-SPIB and low-BATF or the reciprocal low-SPIB and high-BATF mRNA expression links to differential gene expression profiles across nine data sets, identifying distinct associations with SPIB occupancy, signatures of B-cell differentiation stage and potential pathogenetic mechanisms. In a population-based patient cohort, SPIBhigh/BATFlow-ABC-DLBCL is enriched for mutation of MYD88, and SPIBhigh/BATFlow-ABC-DLBCL with MYD88-L265P mutation identifies a small subgroup of patients among this otherwise aggressive disease subgroup with distinct favourable outcome. We conclude that differential expression of IRF4 cofactors SPIB and BATF identifies biologically and clinically significant heterogeneity among ABC-DLBCL.

Plasmablasts as Migratory IgG-Producing Cells in the Pathogenesis of Neuromyelitis Optica

Neuromyelitis optica (NMO) is an inflammatory disease characterized by recurrent attacks of optic neuritis and myelitis. It is generally accepted that autoantibodies against aquaporin 4 water channel protein play a pathogenic role in neuromyelitis optica. We have recently reported that plasmablasts are increased in the peripheral blood of this autoimmune disease, and are capable of producing autoantibodies against aquaporin 4. Here, we demonstrate that CD138+HLA-DR+ plasmablasts, a subset of IgG-producing cells, are increased in the peripheral blood and are enriched among the cerebrospinal fluid (CSF) lymphocytes during the relapse of neuromyelitis optica. Notably, these CD138+HLA-DR+ plasmablasts overexpress CXCR3, whose ligands are present in the cerebrospinal fluid during the relapse of neuromyelitis optica. These results led us to speculate that plasmablasts producing anti-aquaporin 4 autoantibodies might traffic toward the central nervous system (CNS). Furthermore, we performed single-cell sorting of plasmablasts from peripheral blood and CSF samples from NMO and sequenced the complementarity-determining regions (CDRs) of the IgG heavy chain expressed by the sorted plasmablast clones. There were high frequencies of mutations in the CDRs compared with framework regions, indicating that these plasmablast clones would represent a post-germinal center B-cell lineage. Consistent with the preceding results, the plasmablast clones from the peripheral blood shared the same CDR sequences with the clones from the CSF. These results indicate that IgG-producing plasmablasts, which are guided by helper T-cells, may migrate from the peripheral blood preferentially to the CSF. Since migratory plasmablasts could be involved in the inflammatory pathology of NMO, the B-cell subset and their migration might be an attractive therapeutic target.

Genomic Instability: The Driving Force behind Refractory/Relapsing Hodgkin’s Lymphoma

In classical Hodgkin’s lymphoma (HL) the malignant mononuclear Hodgkin (H) and multinuclear, diagnostic Reed-Sternberg (RS) cells are rare and generally make up <3% of the total cellular mass of the affected lymph nodes. During recent years, the introduction of laser micro-dissection techniques at the single cell level has substantially improved our understanding of the molecular pathogenesis of HL. Gene expression profiling, comparative genomic hybridization analysis, micro-RNA expression profiling and viral oncogene sequencing have deepened our knowledge of numerous facets of H- and RS-cell gene expression deregulation. The question remains whether disturbed signaling pathways and deregulated transcription factors are at the origin of refractory/relapsing Hodgkin’s lymphoma or whether these hallmarks are at least partially related to another major factor. We recently showed that the 3D nuclear organization of telomeres and chromosomes marked the transition from H- to RS-cells in HL cell lines. This transition is associated with progression of telomere dysfunction, shelterin disruption and progression of complex chromosomal rearrangements. We reported analogous findings in refractory/relapsing HL and identified the shelterin proteins TRF1, TRF2 and POT1 as targets of the LMP1 oncogene in post-germinal center B-cells. Here we summarize our findings, including data not previously published, and propose a model in which progressive disruption of nuclear integrity, a form of genomic instability, is the key-player in refractory/relapsing HL. Therapeutic approaches should take these findings into account.

EBV-positive diffuse large B-cell lymphoma of the elderly is an aggressive post-germinal center B-cell neoplasm characterized by prominent nuclear factor-kB activation

AbstractHere, we report a retrospective series of 47 EBV-positive diffuse large B-cell lymphoma associated with advanced age. Histopathology allowed to the identification of different histological patterns: cases with polymorphic diffuse large B-cell lymphoma (29 cases), Hodgkin-like (8 cases) and polymorphic lymphoproliferative disorder-like (9 cases) patterns. One case was purely monomorphic diffuse large B-cell lymphoma. We show that this lymphoma type is a neoplasm with prominent classical and alternative nuclear factor-kB pathway activation in neoplastic cells (79% of the cases showed nuclear staining for p105/p50, 74% for p100/p52 and 63% for both proteins), with higher frequency than that observed in a control series of EBV-negative diffuse large B-cell lymphoma (χ2 <0.001). Most cases showed an activated phenotype (95% non-germinal center (Hans algorithm); 78% activated B cell (Choi algorithm)). Clonality testing demonstrated IgH and/or K/Kde/L monoclonal rearrangements in 64% of cases and clonal T-cell populations in 24% of cases. C-MYC (1 case), BCL6 (2 cases) or IgH (3 cases) translocations were detected by FISH in 18% cases. These tumors had a poor overall survival and progression-free survival (the estimated 2-year overall survival was 40±10% and the estimated 2-year progression-free survival was 36±9%). Thus, alternative therapies, based on the tumor biology, need to be tested in patients with EBV-positive diffuse large B-cell lymphoma of the elderly.

Quantitative Proteomic Profiling Unravels Dynamic Changes in the Myeloma Cell Proteome Treated with Valproic Acid (VPA)

Abstract 1847Multiple Myeloma (MM) is a post germinal center B-cell malignancy characterized by abnormal proliferation of terminally differentiated plasma cells in the bone marrow. Despite significant advances in multiple myeloma treatment, the disease remains incurable. Earlier studies have shown that HDAC inhibitors are effective in inducing antitumor effects in many hematological malignancies, but the exact mechanism through which they act is unclear. Since Valproic acid (a FDA approved HDAC inhibitor to treat epilepsy) is cost effective and suitable for old patients, we investigated VPA in this study. Our study aims to unravel the mechanism by which VPA induces antitumor effects in MM cell lines using both biochemical and proteomics approaches.In our experiments, MM cell lines (RPMI8226, NCI H929 and MM1R) when treated with VPA showed a block in the G1-S transition (decrease in the S-phase and G2M-phase, increase in the Sub-G0 phase) and decrease in the cell viability in a dose and time dependent fashion. Also, Annexin V staining by FACS and PARP cleavage by immunoblot analysis confirmed the induction of apoptosis through Caspases. We then analyzed the expression pattern of several genes, including those involved in transcription, cell cycle regulation and signaling pathway by quantitative RT PCR and by immunoblot analysis. Our study showed an induction of H3 acetylation, p21 and an increase in the NOTCH1 target genes (Hes1 and Hey1) in the myeloma cells treated with VPA.To further understand the key signaling pathways involved in the pathogenesis of MM and also to characterize how VPA regulates growth arrest, we investigated the differentially expressed proteome in the RPMI 8226 cell line treated with and without VPA using SILAC (Stable Isotope Labelling with Amino acids in Cell culture) based quantitative proteomics approach. Briefly, one population of RPMI 8226 cells was grown in medium with heavy (isotopic) amino acids (13C6 L-Lysine and 13C6 L-Arginine), while the other population was grown in medium containing naturally abundant isotopic form of (normal) amino acids (12C6 L-Lysine and 12C6 L-Arginine). Cells grown in heavy medium were left untreated, while the cells grown in light medium were treated with 1mM VPA for 24 hrs. Cell lysates were pooled, resolved on SDS-PAGE, protein bands excised, trypsin digested and analyzed on LTQ orbitrap velos mass spectrometer.Using SILAC approach, we identified and quantified 2,032 proteins in myeloma cells treated with VPA. We found that several proteins including protein kinases, Cell cycle regulators, transcription factors, membrane proteins, mitochondrial proteins and metabolic enzymes were regulated by VPA. Checking for the presence of known cell surface markers for plasma cell in the proteome data, we found Syndecan (CD138) with 3 unique peptides and noted that its expression was decreased upon VPA treatment. This was confirmed by FACS analysis. Interestingly, we also found that CCND2, an important regulator of plasma cell development and the one that is often implicated in myeloma pathogenesis, was significantly down regulated by VPA treatment. This was confirmed by Immunoblot analysis in a dose and time dependent manner. We also found that VPA is more effective in regulating CCND2 promoter activity in combination with Dexamethazone.Based on the above results, we then reasoned if NOTCH1 induction could regulate CCND2 expression. Interestingly, our study showed that active form of intracellular NOTCH1 down regulated CCND2 promoter activity. NOTCH1 being a membrane bound transcriptional activator, we then hypothesized that increased NOTCH1 signaling pathway could down regulate CCND2 expression by inducing a transcriptional repressor. Our preliminary results showed that NOTCH1 mediated Hes1 induction repressed the promoter activity of CCND2.Overall, our global quantitative proteomic analysis demonstrates that Valproic acid treatment induces dynamic changes in the myeloma proteome. In addition, we have shown that VPA may control the proliferation of myeloma cells at least in part via a NOTCH-Hes1-CCND2 regulatory axis. These results provide an invaluable starting point to design and use Valproic acid in combination with Dexamethazone and/or with Bortezomib as an effective therapy for myeloma. Disclosures:No relevant conflicts of interest to declare.

Intravascular large B‐cell lymphoma with cutaneous manifestations: a clinicopathologic, immunophenotypic and molecular study of three cases

Intravascular large B-cell lymphoma (IVLBCL) is an extremely rare type of malignant lymphoma characterized by exclusive or predominant growth of neoplastic cells within the lumen of blood vessels. Cases in the literature predominantly involve the skin and central nervous system, with special emphasis on the 'cutaneous variant'. Three cases of IVLBCL with cutaneous manifestations, including two systemic IVLBCL and one cutaneous variant, were described in this study. In all cases, clinical presentation and follow-up data were meticulously evaluated and immunophenotypic and molecular studies were performed. All three cases displayed the B-cell phenotype and showed monoclonality with immunoglobulin heavy chain gene rearrangement. Bcl2 was expressed in the two systemic IVLBCL cases with fatal outcomes. The third patient with the 'cutaneous variant' achieved complete remission and a longer survival time of 15 months after chemotherapy. Skin manifestations and neurological findings, although to different degrees, are important clues to the diagnosis of IVLBCL. As most IVLBCL are grouped into the post-germinal center B-cell subtype of diffuse large B-cell lymphoma, Bcl2 expression may be correlated with a worse prognosis in IVLBCL. The cutaneous variant of IVLBCL has a significantly better outcome than that of systemic IVLBCL.

Pathophysiology of Retinal Lymphoma

Retinal lymphoma, the most common form of intraocular lymphoma, is a high-grade malignancy, usually of B-cell type, and is associated with a poor prognosis because of frequent central nervous system (CNS) involvement. The neoplastic B-cells of retinal lymphoma have a characteristic morphology and immunophenotype, express certain chemokines and chemokine receptors, and produce interleukins (IL), e.g. IL-10. Together with the cytological features of these tumors, the immunophenotype, presence of immunoglobulin rearrangements, and biochemical profile aid the diagnosis of retinal lymphomas. Immunophenotyping and somatic mutation analysis suggest derivation of most retinal lymphomas from an early post-germinal centre B-cell. Chromosomal translocation data would suggest, however, that a subgroup of these neoplasms may arise from germinal centre B-cells, and these could be associated with a better prognosis. Further investigations, such as gene expression profiling, are required to identify oncogenic pathways potentially involved in retinal lymphoma development, and to identify new prognostic/therapeutic markers for this tumor.

Aberrant Co-Expression of Key Transcription Factors BCL6, IRF4 and FOXP1 Are Frequently Observed in Diffuse Large B-Cell Lymphoma

Diffuse Large B-cell Lymphoma (DLBCL) is classified into germinal centre (GCB) and activated B-cell (ABC) type by comparison with the phenotype of normal B-cells. Using single-color immunocytochemistry, tumors can be classified using a simple algorithm based on the expression of CD10, BCL6 and IRF4. This classification may have prognostic relevance and correlates with balanced translocations involving the immunoglobulin locus. However, the phenotypic differences between normal and neoplastic cells may be of greater relevance to understanding the pathogenesis of DLBCL and in developing effective diagnostic techniques. To investigate this we examined the co-expression of BCL6, IRF4 and FOXP1. These are key transcription factors that regulate the process of germinal centre and post germinal centre B-cell differentiation. Abnormal co-expression of these molecules would be expected to have major effects on the overall cellular phenotype. A multi-color immunofluorescence (MCIF) technique was developed that allowed the co-expression of these markers to be assessed in relation to the PAX5 positive B-cell population. The use of a multi-color technique allows the distinction between co-expression at the level of individual cells and differentiation within the tumor as a whole. We first determined the pattern of expression of these transcription factors in normal B-cells. In reactive lymph nodes the expression of BCL6, IRF4 and FOXP1 was almost mutually exclusive with only a small proportion of co-expressing cells. In a series of 61 DLBCL co-expression of both BCL6/IRF4 and IRF4/FOXP1 was found in 41/61 (67%) of the cases. In most of these cases the level of co-expression was greater than 50% of the PAX5 positive large lymphoid cells. Co-expression was not present in 11/61 (18%) of the tumors. In the remaining cases there was co-expression of either BCL6/IRF4 or IRF4/FOXP1. There was no correlation between the occurrence of co-expression of these combinations of transcription factors and the expression of CD10 or the classification into GCB and ABC phenotypes. In 16 of the cases the sample used was a small needle core biopsy in which assessment of nodal architecture was impossible. In these cases it was possible to confidently determine the presence of abnormal co-expression in 14/16 (87.5%) of the cases. One explanation for the aberrant co-expression of BCL6 and IRF4 in DLBCL would be the presence of a 3q27 rearrangement leading to dysregulation of BCL6 expression. However, in this series there was no correlation between BCL6/IRF4 co-expression and abnormalities of 3q27 detected by interphase FISH. These results show that in the majority of cases of DLBCL the key transcription factors regulating post germinal centre B-cell differentiation are expressed in combinations not seen in normal B-cells. This is likely to be a central element in the pathogenesis of these tumors. The ability to reliably identify these abnormalities by MCIF has potential value in improving the reliability of diagnosis of DLBCL when only small biopsy samples are available and it is likely that this approach can be extended to other types of lymphoma.

409 POSTER A Phase I clinical trial of the oral PPAR gamma agonist, CS-7017 in patients with advanced malignancies

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

Hybridoma populations enriched for affinity-matured human IgGs yield high-affinity antibodies specific for botulinum neurotoxins

The affinity-matured human antibody repertoire may be ideal as a source for antibody therapeutics against infectious diseases and bioterror agents. Hybridoma methods for cloning these antibodies have many potential advantages, including convenience, high-yield antibody expression, and the ability to capture the antibodies in their native configurations. However, they have been hindered by hybridoma instability and limited accessibility of antigen-specific, class-switched human B-cells. Here, we describe an efficient, three-step method that uses human peripheral blood B-cells to produce stable hybridoma populations that are highly-enriched for affinity-matured human IgG antibodies. Peripheral blood mononuclear cells (PBMCs) are (a) selected for expression of CD27, a marker of post-germinal center B-cells, (b) cultured in vitro to promote B-cell proliferation and class-switching, and (c) fused to a genetically modified myeloma cell line. Using this strategy, we cloned 5 IgG antibodies that bind botulinum neurotoxins (BoNT), the causes of the food-borne paralytic illness, botulism, and Category A Select Bioterror agents. Two of these antibodies bind BoNT with low picomolar affinities. One (30B) is the first high-affinity human antibody to bind serotype B BoNT, and another (6A) is able to neutralize a lethal dose of serotype A BoNT in vivo in pre- and post-exposure models. This optimized hybridoma method will broadly enable access to the native human antibody repertoire.

Bcl‐6 predicts improved prognosis in primary central nervous system lymphoma

In systemic non-Hodgkin lymphoma (NHL), tumors that resemble germinal center B-cells are less aggressive than tumors that resemble postgerminal center B-cells. However, the value of B-cell differentiation markers in primary central nervous system lymphoma (PCNSL) is less clear. The objectives of this study were to characterize the immunophenotypes of PCNSL samples and to determine their utility in predicting clinical outcomes. The immunohistochemical staining profile of PCNSL was determined from 66 immunocompetent patients. Then, the authors evaluated whether the expression of these proteins was associated with progression-free or overall survival. Only 8% of PCNSL samples were positive for the cluster designation (CD) germinal center marker CD10. Another germinal center marker, bcl-6, was positive in 47% of samples. Ninety-four percent of samples expressed significant levels of the postgerminal center marker melanoma-associated antigen (MUM-1). In univariate analyses, only bcl-6 staining had a significant effect on progression-free survival (median, 20.5 months in bcl-6-positive patients vs 10.1 months in bcl-6-negative patients; P = .02). There was a nonsignificant trend toward improved overall survival in patients who had bcl-6 expressing tumors. Older age and poorer performance status, as observed previously, were associated with reduced survival. Bcl-6 expression was associated with a better prognosis in patients with PCNSL.

Plasmablastic Lymphoma of Head and Neck: Report of Two New Cases and Correlation with c-myc and IgVH Gene Mutation Status

Plasmablastic lymphoma (PBL) is a rare acquired immunodeficiency syndrome-associated non-Hodgkin's lymphoma (AIDS-NHL), with predilection for the mucosa of oral cavity. It usually has a plasmablastic morphology, expressing plasma cell-associated antigens with weak or absent expression of B-cell-associated markers. To further define the immunophenotypic and molecular genetics of these tumors, we investigated two cases of plasmablastic lymphomas of the head and neck for c-myc gene rearrangement and immunoglobulin heavy chain (IgV(H)) hypermutation status. For the first time we report a case of AIDS-related PBL that, by fluorescence in situ hybridization (FISH), shows a c-myc gene rearrangement. Although current literature suggests that most cases of c-myc gene rearranged AIDS-NHL are Burkitt's lymphoma, our case has an immunophenotype characteristic for PBL. In this case, IgV(H) hypermutation analysis showed a somatic hypermutation, indicative of germinal center transit. The concurrent B-cell immunophenotype of BCL-6(-)/CD138(+)/MUM-1(+) also suggests a post-germinal center B-cell origin of this lymphoma. The immunophenotype of our second case (BCL-6(-)/CD138(+)/MUM-1(+)) also suggests a post-germinal center B-cell origin. However, IgV(H) hypermutation analysis was not possible in this case.

Profiling of diffuse large B‐cell lymphoma by immunohistochemistry: identification of prognostic subgroups

Diffuse large B-cell lymphoma (DLBCL) is a frequent lymphoma subtype with a heterogeneous behavior and a variable response to conventional chemotherapy. This clinical diversity is believed to reflect differences in the molecular pathways leading to lymphomagenesis. In this study, we have analyzed pretreatment, diagnostic samples from 108 DLBCL by immunohistology for expression of four markers linked to germinal center B-cells (CD10, Bcl-6), postgerminal center B-cells (MUM1) and apoptosis (Bcl-2). The results indicate that both CD10 and Bcl-6 are favorable prognostic indicators, in contrast to Bcl-2, which is an adverse parameter. Furthermore, using two algorithms for distinction between low- and high-risk patients proposed by Hans et al. (Blood, 2004; 103:275) and Muris et al. (Journal of Pathology, 2006; 208:714), it is shown that both are useful for predicting outcome in DLBCL. However, in this report, the algorithm of Hans et al. was superior to that of Muris et al. These findings confirm and extend other studies and indicate that different prognostic subgroups of DLBCL can be distinguished by simple immunohistological investigations for a limited number of markers. Whether these groups are also relevant for individual treatment decisions will be important to investigate in prospective studies.

DA-EPOCH-R Is Highly Effective in Both BCL-6+ and BCL-6− Untreated De Novo Diffuse Large B-Cell Lymphoma (DLBCL): Study Update and Analysis of Survival Outcomes for Multiple Biomarkers.

Gene expression profiling has yielded a new molecular taxonomy of DLBCL in which 3 subtypes are distinguished; germinal center B-cell (GCB) subtype, derived from a germinal center B-cell; activated B-cell (ABC) subtype, derived from a post-germinal center B-cell; and primary mediastinal B-cell (PMBL) subtype, likely derived from a “thymic” B-cell. To define the effect of pathobiology on treatment outcome, immunophenotype has been used as an “approximate surrogate” biomarker for molecular taxonomy. Recent studies suggest that rituximab (R) benefit is primarily in BCL-6− (Winter et al Blood 107: 4207, 2006) and BCL-2+ (Mounier et al Blood 101:4279, 2003) DLBCL, both more frequent in post-germinal center subtypes. Conversely, R-CHOP appears equivalent to CHOP in BCL-6+ DLBCL, which comprise up to 75% of all cases. We analyzed the outcome of DA-EPOCH-R using the immunophenotypic biomarkers BCL-6, BCL-2, MIB-1, CD10, MUM-1, and GCB vs ABC subtypes by the method of (Hans et al Blood 103:275, 2003). Patients had untreated de novo DLBCL excluding PMBL, stage II-IV, HIV-, and adequate organ function. No patients received radiation. Characteristics of 72 patients include median (range) age 50 (19–85); stage III/IV 69%; and High Intermediate/High International Prognostic Index (3–5) (IPI) 40%. Of 71 evaluable patients, response is CR/CRu 94%. At the median potential follow-up of 43 months, PFS is 82% and OS is 79% (Fig 1). PFS by Low/Low Intermediate (0–2) and High Intermediate/High (3–5) IPI is 93% and 64%, respectively, at 43 months. PFS by BCL-6 (Fig 1), and BCL-2 and GCB vs. ABC (Fig 2) is shown. PFS of DLBCL with MIB-1 < and > 80% was 92% and 81%, respectively. There was no significant difference in survival outcome by CD10 or MUM-1 expression (data not shown). Distribution by immunophenotype is shown below for patients with available tissue.Biomarker DistributionBiomarkerNumberPercentBCL-6 Neg1424BCL-6 Pos4476BCL-2 Neg2441BCL-2 Pos3559GCB3467ABC1733MIB-1 < 80%1333MIB-1 > 80%3967CD10 Neg3663CD10 Pos2137Mum-1 Neg3064Mum-1 Pos1736In conclusion, DA-EPOCH-R appears equally effective among all biomarker subgroups. Of note, DA-EPOCH-R is highly effective in BCL-6+ DLBCL, in which R does not appear to be very useful, suggesting the DA-EPOCH regimen itself may be highly effective in this group. The CALGB has initiated a Phase III randomized study of R-CHOP vr. DA-EPOCH-R to determine if DA-EPOCH-R represents a treatment advance. Gene expression profiling will be performed to assess the effect of the new molecular taxonomy and tumor biology on outcome. [Display omitted]

Expression of B-cell transcription factors in primary cutaneous B-cell lymphoma

Expression patterns of eight transcription factors involved in different stages of B-cell development were investigated in a large group of primary cutaneous B-cell lymphomas and compared with expression patterns during normal B-cell development. The following transcription factors were investigated: Pax-5, PU.1, Oct2, BOB.1, Bcl-6, Mum1/IRF4, Blimp-1 and FOXP1. Primary cutaneous large B-cell lymphomas, leg type showed aberrant coexpression of Bcl-6 and Mum1/IRF4 and in addition strong expression of FOXP1. Expression of FOXP1 and Mum1/IRF4 strongly suggests an activated B-cell type of origin. In contrast, primary cutaneous follicle center lymphomas showed expression of Bcl-6, Pax-5, PU.1, Oct2 and BOB.1, but not of Mum1/IRF4, Blimp-1 and FOXP1. Primary cutaneous marginal zone B-cell lymphoma showed expression of Pax-5, PU.1, Oct2 and BOB.1, but not Bcl-6 by the neoplastic B-cells, and Mum1/IRF4 and Blimp-1 by the neoplastic plasma cells. In conclusion, in primary cutaneous follicle center lymphoma and primary cutaneous marginal zone B-cell lymphoma expression patterns were observed similar to their supposed benign counterparts, germinal center B-cells and postgerminal center B-cells, respectively, which might reflect their indolent clinical behaviour and excellent prognosis. In contrast, the activated B-cell expression pattern in the group of primary cutaneous large B-cell lymphoma, leg type may contribute to its poor prognosis and Mum1/IRF4 and FOXP1 may serve as additional diagnostic markers for this type of primary cutaneous B-cell lymphoma.

In Reed Sternberg Cells Suppression of the B-Cell Phenotype and Cell Proliferation Is Not Mediated by BLIMP-1.

Expression of the transcriptional repressor BLIMP-1 is a pivotal event in the differentiation of post germinal centre B-cells to plasma cells. BLIMP-1 extinguishes the B-cell phenotype by down-regulation of PAX-5 with associated loss of BCL-6 and PU. and acting in concert with IRF-4 and XBP-1 induces the expression of the mature plasma cell phenotype. A key step in this process is the permanent exit of the differentiating plasma cell from the cell cycle which appears to be partly dependent on BLIMP-1 mediated repression of c-MYC. Reed Sternberg cells in Classical Hodgkin Lymphoma are known to be derived from post germinal centre B-cell which have undergone extinction of most elements of their B-cell phenotype but do not complete terminal differentiation to plasma cells. The primary aim of this study was to establish whether this loss of B-cell characteristics was mediated by BLIMP-1 and whether this was associated with exit from the cell cycle. 35 well characterised biopsies of nodular sclerosing Classical Hodgkin Lymphoma were studied using an extensive panel of B-cell and cell cycle markers. BLIMP-1 was identified using a BLIMP-1 alpha specific polyclonal antibody raised against the BLIMP-1 amino terminus which was validated by Western Blotting and immunocytochemical reactivity with normal plasma cells and squamous epithelia. All of the Reed Sternberg cells in all of the cases studied had the immunophenotype CD30+, IRF-4+, PU.1-. 15% cases showed PAX-5 a proportion of tumour cells and CD20 and or CD79 were found in 6% of cases. In 21% cases there was either nuclear or cytoplasmic expression of C/EBP alpha. All Reed Sternberg cells expressed Ki67 and around 50% of cells in all of the cases expressed the G2/M phase marker geminin with p53 and p21 uniformly expressed in the tumour cells. In contrast to the patterns seen in normal B-cell differentiation there was no evidence of BLIMP-1 or XBP-1 expression in any of the cases studied. To further clarify the reason for cell cycle arrest, interphase FISH using a panel of centromeric probes was performed on imprints preparations of lymph nodes replaced by Hodgkin lymphoma. This showed asymmetrical chromosome separation between the nuclear lobes of individual Reed Sternberg cells and heterogeneity of chromosome content within the mononuclear tumour cell fraction. These results indicate that extinction of the B-cell phenotype in Reed Sternberg cell is not dependent on the induction of BLIMP-1expression and that unlike B-cells undergoing differentiation to plasma cells most Reed Sternberg cells undergo cell cycle arrest mediated by normal p53 expression rather than permanently exiting from the cell cycle. Cell cycle arrest is likely to be related to asymmetrical chromosomal segregation within the tumour cell population. Previous experimental studies have shown that plasma cells differentiation is blocked in cells that are continuing to cycle. The results of this study raise the possibility that cell cycle arrest due to abberrant mitosis may be a key factor in determing the distinctive phenotype of Reed Sternberg cells through a mechansim that is independent of BLIMP-1 activation.

Characterization of Patients with Multiple Myeloma and t(4;14) Detected by Fluorescence In Situ Hybridization.

Multiple myeloma (MM) is a genetically unstable malignancy of postgerminal center B-cells. Almost 40% of intramedullary MM tumors show primary translocations that affect immunoglobulin heavy chain (IgH) gene. These include t(4;14)(p16;q32) which results in the dysregulated expression of two potential oncogenes, MMSET on der(4) and FGFR3 on der(14), and has been associated with poor outcome. The main goals of this study were to determine the incidence and clinical significance of t(4;14) among our MM patients. Therefore, we studied bone marrow specimens from 65 patients with MM by fluorescence in situ hybridization (FISH). All cases were screened for IgH rearrangements, t(4;14), t(11;14), and 13q14 deletions using the locus-specific probes LSI IgH dual color, break apart, LSI IGH/FGFR3, LSI IGH/CCND1, and LSI D13S319 (Vysis). FISH analysis revealed 35 cases (54%) involving IgH locus. 8 patients (12.3%) had a t(4;14), and 13 cases (20%) showed a t(11;14). In the remaining 14 samples (21.5%), IgH rearrangements were observed, but the translocation partner was not one of the loci for we tested. Furthermore, 13q14 deletions were more frequent among patients with t(4;14) than among those with t(11;14) (62.5% vs 30.7% respectively, p=0.03). Regarding clinical parameters, presence of t(4;14) was significantly associated with anemia (mean value: 9.0 g/dl, p=0.049), elevated LDH levels (mean value: 535.4 U/l, p=0.05), Durie III stage (p=0.049), and number of lytic bone lesions >2 (p=0.007). Finally, the survival median of patients with t(4;14) was 23 months vs 48 months for the group without this abnormality. Cases with t(11;14) did not show adverse correlation with survival.Based on these data, FISH is a successful technique to detect translocations affecting telomeric localization of both chromosomal partner loci. In addition, we confirmed t(4;14) as an important factor of poor prognostic in MM. Its detection is essential to lead a correct evaluation of patients at diagnosis.

MUM1 Expression in Follicular Lymphoma Identifies a Group of Patients with Poor Overall Survival.

Background: Follicular lymphoma (FL) is the second most common type of lymphoma in the United States. The clinical course is variable--some patients have an indolent course while others experience relatively aggressive disease, transformation to higher grade, and short survival. Despite a wide range of treatment options, no consensus exists concerning optimal therapy. Clinical predictors of outcome such as the FL International Prognostic Index (FLIPI) exist; however, biologic risk factors are needed to assist in providing accurate risk stratification. Recent data have suggested that the anti-tumor immune response may affect the clinical course and survival. In this study we sought to identify biologic indicators of prognosis in FL patients using a tissue microarray.Methods: Biopsies from 94 newly diagnosed FL patients who presented between 1985 and 2002 were reviewed for cytologic grade (CG)(Mann-Berard criteria, WHO classification) and presence of diffuse areas (DAs) prior to placement in a tissue microarray (two 1.5 mm cores/case). Cases with diffuse large B-cell components were not included. Immune response was assessed with immunostains for CD3 and CD25 as indices of T-cell infiltration and activation, respectively, and CD68 and CD163 to evaluate macrophage infiltration. Lymphoma cells were evaluated for bcl-2 expression, as well as CD10 and MUM1 expression to assess germinal center phenotype. Initial and subsequent patient management was individualized and varied considerably. Clinical data were obtained for FLIPI and overall survival (OS). Statistical analysis was performed using P ≤ 0.10 as significant for univariable and P ≤ 0.05 for multivariable analysis.Results: The patients consisted of 50 males (53.2%) and 44 females (46.8%), with a median age at diagnosis of 58 (range 24–89) years. The FLIPI distribution was 34 low risk (0–1), 26 intermediate risk (2), and 34 high risk (>2). There were 31 deaths. The median survival was 174.1 months and the median follow-up among surviving patients was 68.8 months (range 19.5–196.3). Cox proportional hazards analysis for risk of death showed no association with CG, DAs, or expression of bcl-2, CD3 (intrafollicular), CD25, CD68, or CD163. A high FLIPI score and lack of CD10 expression were both of borderline significance for higher risk of death (HR 2.06, 95% CI 0.86–4.92; P = 0.10 and HR 2.8, 95% CI 0.81–9.65; P = 0.10, respectively). MUM1 was expressed in 23 of 87 evaluable cases and was also associated with higher risk of death (HR 2.14, 95% CI 0.97–4.71; P = 0.059). Multivariable analysis showed that only MUM1 expression was associated with a higher risk of death (HR 2.30, 95% CI 1.04–5.12; P = 0.04).Conclusions: These data demonstrate that expression of MUM1 in FL cells, consistent with a late germinal center/post germinal center phenotype, identifies a group of FL patients with poor prognosis. We found no correlation between survival and host immune response, using individual analysis of immunohistochemical markers of T-cell infiltration/activation (CD3 and CD25) or macrophage infiltration (CD68 and CD163). More detailed analysis of molecules involved in the transition of centrocyte to post-germinal center B-cells in FL is warranted.

Hodgkin’s disease variant of Richter’s syndrome clonally related to chronic lymphocytic leukemia arises in ZAP-70 negative mutated CLL

Chronic lymphocytic leukemia (CLL) may derive from either immunoglobulin V(H) gene unmutated (naïve) or mutated (antigen-experienced) post-germinal center B-cells. Richter's syndrome denotes the transformation of CLL into aggressive B-cell lymphoma. Most Richter's syndrome cases are secondary diffuse large B-cell lymphomas, but some are Hodgkin's disease variants. Hodgkin's lymphoma is thought to originate from germinal center or post-germinal center B-cells with evidence of somatic V(H) hypermutation. Taking into account CLL and Hodgkin's lymphoma histogenesis, hypothetically only CLL derived from V(H) mutated B-cells can clonally progress to Hodgkin's lymphoma variant of Richter's syndrome. To test our hypothesis, we analyzed the CLL ZAP-70 status as a surrogate for the V(H) mutational status in four patients with subsequent Hodgkin's disease variants of Richter's syndrome. In all three cases with proven or suspected clonal relationship between Hodgkin and Reed-Sternberg- and leukemia cells, the CLL samples remained negative for ZAP-70, corresponding to CLL histogenesis from V(H) mutated B-cells. These empirical data suggest that the histological and clinical diversity of Richter's transformation could be to a part explained by the different origin of CLL, with Hodgkin's disease variants arising probably only in V(H) mutated CLL.