Human Germinal Center-associated Lymphoma Research Articles

Role of an adaptor protein human germinal center-associated lymphoma (HGAL) in cell signaling and lymphomagenesis

Human germinal center (GC)-associated lymphoma (HGAL) is a multi-domain adaptor protein expressed in GC B lymphocytes, T follicular helper (Tfh) cells and lymphomas derived from these cells. HGAL expression is an independent predictor of longer survival of diffuse large B-cell lymphoma (DLBCL) and classical Hodgkin’s lymphoma (HL) patients. HGAL regulates B cell receptor (BCR) signaling and immunological synapse formation by binding to either the downstream effectors [e.g., spleen tyrosine kinase (Syk)] or other signaling regulators [e.g., growth factor receptor-bound protein 2 (Grb2)]. HGAL regulates the cytoskeleton that reshapes B cell morphology during BCR signaling and cell motility by at least two molecular mechanisms: enhanced Ras homolog gene family member A (RhoA) signaling and inhibition of myosin-actin translocation. These effects on the cytoskeleton decrease lymphoma dissemination in animal models and contribute to decreased lymphoma dissemination in patients. The latter may contribute to the association of HGAL protein expression with longer survival of patients with DLBCL and HL tumors. The ability to regulate multiple and distinct functions simultaneously in B cells implies that the HGAL protein level is tightly regulated. It was demonstrated that HGAL can be regulated by PR/SET domain 1 (PRDM1)/B lymphocyte-induced maturation protein-1 (BLIMP1) and interleukin-4 (IL-4) at the transcription level, by microRNA-155 (miR-155) at the post-transcriptional level, and by F-box protein 10 (FBXO10) at the post-translational level. Constitutive enforced expression of HGAL at physiological levels leads to lymphoid hyperplasia and DLBCL in mice. Future studies need to focus on identifying HGAL interactome, dissecting its interaction network, and understanding HGAL spatiotemporal signaling in live cells in physiological conditions. Further, the recent demonstration of HGAL expression in Tfh cells requires the determination of its function in these cells. These studies will contribute to new insights into the biology of these cellular subsets and how immune dysregulation contributes to lymphomagenesis.

Human Germinal Center-associated Lymphoma (HGAL) Is a Reliable Marker of Normal and Neoplastic Follicular Helper T Cells Including Angioimmunoblastic T-Cell Lymphoma.

The diagnosis of angioimmunoblastic T-cell lymphoma (AITL) is complex and requires the demonstration of a T-follicular helper (TFH) phenotype. Immunophenotypic markers that detect the TFH phenotype are highly variable, thereby necessitating the use of 3 to 5 TFH markers to substantiate a TFH phenotype. We tested the utility of germinal center markers human germinal center-associated lymphoma (HGAL) and LIM-domain only 2 (LMO2) in detecting a TFH phenotype. We compared their staining to that of 6 TFH markers in current use, PD-1, ICOS, CXCL13, SAP, CD10, and BCL6, in a cohort of 23 AITL. Our results show that although both markers can detect a TFH phenotype, HGAL was superior to LMO2 in the percent of cells stained and the intensity of staining, 2 variables used to generate H-scores. Using H-scores as the metric, HGAL was most comparable to BCL6 among the currently used TFH markers and was more sensitive than CXCL13, SAP, CD10, and LMO2. PD-1 and ICOS emerged as the most robust of the 8 markers tested in this study in detecting a TFH phenotype. We conclude that HGAL is a reliable marker of TFH cells and can aid in the diagnosis of lymphomas of TFH derivation, particularly in the recognition of early patterns of AITL.

Conditional expression of HGAL leads to the development of diffuse large B-cell lymphoma in mice

Diffuse large B-cell lymphomas (DLBCLs) are clinically and genetically heterogeneous tumors. Deregulation of diverse biological processes specific to B cells, such as B-cell receptor (BCR) signaling and motility regulation, contribute to lymphomagenesis. Human germinal center associated lymphoma (HGAL) is a B-cell–specific adaptor protein controlling BCR signaling and B lymphocyte motility. In normal B cells, it is expressed in germinal center (GC) B lymphocytes and promptly downregulated upon further differentiation. The majority of DLBCL tumors, primarily GC B-cell types, but also activated types, express HGAL. To investigate the consequences of constitutive expression of HGAL in vivo, we generated mice that conditionally express human HGAL at different stages of hematopoietic development using 3 restricted Cre-mediated approaches to initiate expression of HGAL in hematopoietic stem cells, pro-B cells, or GC B cells. Following immune stimulation, we observed larger GCs in mice in which HGAL expression was initiated in GC B cells. All 3 mouse strains developed DLBCL at a frequency of 12% to 30% starting at age 13 months, leading to shorter survival. Immunohistochemical studies showed that all analyzed tumors were of the GC B-cell type. Exon sequencing revealed mutations reported in human DLBCL. Our data demonstrate that constitutive enforced expression of HGAL leads to DLBCL development.

The molecular landscape and other distinctive features of primary cutaneous follicle center lymphoma

Primary cutaneous follicle center lymphoma (PCFCL) is distinguished from other follicular lymphomas (FLs) based on its clinicopathologic features including diminished CD10 and frequent lack of BCL2 rearrangements (R). Whether newer germinal center-associated markers would also be less commonly expressed and whether mutational studies would support its segregation from classic FL and FL subsets, including those which also typically lack BCL2R, are uncertain. To address these questions, 22 PCFCLs were stained for myocyte enhancer factor 2B (MEF2B) and human germinal center-associated lymphoma (HGAL), and targeted next-generation sequencing was performed with results compared to a meta-analysis of FL, pediatric-type FL (PTFL), low stage FL (LSFL) and other FL subsets. Selected fluorescence in situ hybridization studies were also performed. Although 27% of cases lacked CD10, all tested were MEF2B+ and HGAL+. The most common somatic mutations in the 12 to 19 analyzable PCFCL were TNFRSF14 (40%, plus 10% with 1p36 deletions), followed by CREBBP, TNFAIP3, KMT2D, SOCS1, EP300, STAT6, and FOXO1 (17-25%). Three of the most commonly mutated genes in FL (KMT2D, CREBBP, and BCL2) were significantly less commonly mutated in PCFCL than in FL, and TNFAIP was more commonly mutated with no difference for TNFRSF14 between PCFCL and FL or PTFL. CREBBP was also less frequently mutated than in LSFL but more frequently mutated than in PTFL. MAP2K1 mutations were much more common in PTFL (44% versus 0%). Two of 22 of the PCFCL had a BCL2 rearrangement and zero of 12 had a BCL6 rearrangement. These findings, while showing well-recognized and new shared features between PCFCL and other FL, highlight a distinctive mutational profile further supporting its recognition as a distinct entity.

Recent BCR stimulation induces a negative autoregulatory loop via FBXO10 mediated degradation of HGAL.

Regulating B-cell receptor (BCR) signaling after antigenic stimulation is essential to properly control immune responses. Currently known mechanisms of inhibiting BCR signaling are via co-receptor stimulation and downstream immunoreceptor tyrosine-based inhibition motif (ITIM) phosphorylation. Herein we demonstrate that BCR stimulation induces rapid and reversible palmitoylation of the SCF-FBXO10 ubiquitin E3 ligase. This results in FBXO10 relocation to the cell membrane, where it targets the human germinal center-associated lymphoma (HGAL) protein for ubiquitylation and degradation, leading to decreases in both BCR-induced calcium influx and phosphorylation of proximal BCR effectors. Importantly, FBXO10 recognition and degradation of HGAL is phosphorylation independent and instead relies on a single evolutionarily conserved HGAL amino acid residue (H91) and FBXO10 relocalization to the cytoplasmic membrane. Together our findings demonstrate the first evidence of negative BCR signaling regulation from direct BCR stimulation and define the temporospatial functions of the FBXO10-HGAL axis. FBXO10 is infrequently mutated in DLBCL but some of these mutations deregulate BCR signaling. These observations may have important implications on lymphomagenesis and other immune processes.

The TGFβ-induced lncRNA TBILA promotes non-small cell lung cancer progression in vitro and in vivo via cis-regulating HGAL and activating S100A7/JAB1 signaling

Long non-coding RNAs (lncRNAs) play critical roles in multiple cellular processes in non-small cell lung cancer (NSCLC); however, the involvement of lncRNAs in the transforming growth factor-beta (TGFβ) signaling pathway, the critical tumor cell epithelial-mesenchymal transition (EMT) and metastasis pathway, remains poorly understood. To address this issue, we compared the lncRNAs expression patterns of NSCLC cells treated with and without TGFβ1 treatment. We observed that one of the most prominent hits, TGFβ-induced lncRNA (TBILA), promoted NSCLC progression and was upregulated in tumor tissues. Upregulated TBILA promotes human germinal center-associated lymphoma (HGAL) expression by binding to the Smad transcription factor complex, thereby enhancing RhoA activation. In addition, TBILA induces the S100A7-c-Jun activation domain-binding protein 1 (JAB1) pathway by binding to nuclear S100A7 and enhances pro-survival pathways in NSCLC. These findings have provided us with a new perspective regarding the regulation of the TGFβ signaling pathway in NSCLC and suggest that the lncRNA TBILA can serve as a target for anticancer therapies.

FBXO10 Targets HGAL for Degradation

Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by a better prognosis. We previously showed that HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for successful completion of the GC reaction. In our previous studies we demonstrated that upon BCR activation HGAL binds to and increases Syk kinase activity, resulting in enhanced BCR signaling. Syk induces HGAL phosphorylation, leading to its redistribution from lipid rafts to the bulk membrane and cytoplasm, followed by its rapid degradation. The mechanism of HGAL degradation is currently unknown.To elucidate the mechanism of HGAL degradation, we hypothesized that HGAL is degraded by a SKP1-CUL1-F-box protein (SCF) ubiquitin ligase complex. Consequently, we examined the ability of HGAL to be recruited to the SCF by screening a panel of F-box proteins transfected into HEK293T cells by reciprocal coimmunoprecipitations (Co-IPs). These studies revealed that HGAL specifically bound only FBXO10. Concordantly, HGAL Co-IPed with endogenous SKP1 and CUL1 in the presence of FBXO10. Furthermore, a dose dependent reduction of HGAL levels was observed in Raji cells transfected with increasing quantities of FBXO10 expressing plasmid. In contrast, none of the other FBXO11 tested F-box proteins affected HGAL levels. The reduction in HGAL protein level by FBXO10 was due to enhanced proteolysis, as demonstrated by the decrease in HGAL protein half-life and the rescue of its levels by either addition of proteasome inhibitor MG132 or expression of a dominant-negative CUL1 mutant (CUL1 (1-242)). Furthermore, depletion of FBXO10 by two short hairpin RNA (shRNA) constructs in Raji cells increased HGAL stability and half-life.SCF complexes mediates protein degradation by polyubiquitination. We therefore examined if HGAL protein can be ubiquinated. To this end we cotransfected 293T cells with HGAL-V5, FBXO10-FLAG, HA-ubiquitin with or without CUL1 (1-242) and blotted immunoprecipitated HGAL for ubiquitin. FBXO10 expression markedly increased HGAL ubiquitination and this was significantly decreased in cells coexpressing CUL1 (1-242). Similarly, HGAL ubiquitination was markedly decreased in cells expressing FBXO10 (FBXO10- ΔF-box), in which the F-box motif, necessary for linking the target protein to the SCF ubiquitin ligase moieties, is deleted. These results support the function of FBXO10 in binding HGAL and facilitating its ubiquitination by the SCF complex prior to its degradation.We next analyzed the functional significance of HGAL degradation by FBXO10 on BCR activation, as measured by intracellular and transmembrane Ca2+ mobilization. Overexpression of FBXO10 in Raji cells expressing endogenous HGAL led to decreased levels of HGAL and in Ca2+ mobilization. In contrast, FBXO10 did not affect BCR-induced Ca2+ mobilization in Raji cells in which HGAL was deleted by crispr/Cas9 targeting. These results suggest that FBXO10 affects BCR signaling via regulating HGAL levels.While FBXO10 mediates HGAL degradation post BCR-induced HGAL phosphorylation, FBXO10 also bound HGAL in the absence of BCR activation. The binding of FBXO10 to HGAL was unaffected in Raji cells incubated with anti-IgM antibodies. Treatment of cellular lysates with l-phosphatase did not affect FBXO10 binding to HGAL. These findings indicate that FBXO10-dependent degradation of HGAL is phosphorylation independent. In contrast, HGAL degradation by FBXO10 was dependent on HGAL lipid raft localization, since HGAL myristoylation and palmitoylation mutants (G2A, C43A/C45A, and G2A/C43A/C45A) exhibited decreased Co-IP with the FBXO10 protein. To further map FBXO10 binding motifs of HGAL, we generated HGAL deletion and mutant constructs and examined their ability to Co-IP with FBXO10. These studies revealed that HGAL amino acids 91-95(HRVLC) mediate HGAL binding to FBXO10.In summary, our results demonstrate that SCFFBXO10 is the ubiquitin ligase for HGAL and regulates BCR signaling via controlling HGAL expression. Some diffuse large cell lymphomas expressing HGAL harbor inactivating mutations or express low levels of FBXO10, thus predisposing to enhanced BCR signaling- a phenomenon implicated in lymphomagenesis. DisclosuresNo relevant conflicts of interest to declare.

Grb2 Directly Interacts with HGAL and Ameliorates Its Effects on B-Cell Receptor Signaling

Human Germinal center Associated Lymphoma (HGAL) is specifically expressed in germinal center (GC) B-cells and GC-derived lymphomas. High expression of HGAL is an independent predictor of prolonged survival of Diffuse Large B-Cell (DLBCL) and classical Hodgkin (cHL) lymphoma patients. HGAL is a unique adaptor protein that regulates both cell motility and B-cell receptor (BCR) signaling, processes that are central for the successful completion of the GC reaction. HGAL increases BCR signaling by binding to and enhancing Syk kinase activity. However, our previous studies also suggested that other proteins may be involved in HGAL-mediated regulation of BCR signaling.In vitro kinase assays demonstrated that both Syk and Lyn can phosphorylate HGAL. Mass spectrometry (μ LC/MS/MS) demonstrated that these kinases can phosphorylate HGAL's tyrosines Y80, Y86, Y106Y107, Y128 and Y148. The HGAL Y106Y107 comprise a YYENV motif (aa 106-110) similar to the phosphopeptide motif pYXNX frequently used as a binding site to the SH2 domain of Growth Factor Receptor bound protein 2 (Grb2). Grb2 signaling in B cells controls lymphoid follicle organization and the GC reaction. Specifically, Grb2 is an integral component of the BCR signalosome and decreases BCR-induced Ca2+influx. The presence of the phosphorylated YYENV motif in HGAL raised the hypothesis that HGAL-Grb2 interactions may play a role in HGAL -mediated regulation of BCR signaling. To address this possibility, we performed reciprocal coimmunoprecipitations (Co-IPs) of endogenous HGAL and Grb2 in Raji and VAL lymphoma cell lines. These studies demonstrated that HGAL Co-IPs with Grb2. The interaction between these two proteins is dependent on the presence and phosphorylation of tyrosines in the YYENV motif, since an HGAL mutant in which these tyrosines were mutated to phenylalanine (FFENV) failed to Co-IP with Grb2. Isothermal titration calorimetry confirmed that phosphorylated (pYEN) but not unphosphorylated (YEN) HGAL-derived 12-mer peptides bind to the SH2 domain of Grb2 with an affinity of 5µM. GST-Grb2 pull down assays with recombinant Trx-HGAL(FFENV) and Trx-HGAL proteins confirmed that the HGAL-Grb2 interaction is direct and occurs only if the HGAL tyrosines are phosphorylated. Concordantly, addition of phosphatase to cellular lysates decreased the HGAL-Grb2 interaction. Furthermore, CO-IP studies demonstrated that HGAL's interaction with Grb2 increases following BCR stimulation-induced HGAL phosphorylation. Concordantly, confocal microscopy studies demonstrated HGAL-Grb2 colocalization in the cell membrane following BCR signaling activation.We next examined the functional significance of the HGAL-Grb2 interaction on BCR activation as measured by intracellular and transmembrane Ca2+ mobilization and phosphorylation of proximal BCR effectors (Syk (Y352), BLNK (Y84), BTK (Y551) and PLCγ2 (Y753) in several lymphoma cell lines (U2942, TMD8 and Mino) stablly transfected to express HGAL protein. HGAL expression markedly increased Ca2+ influx and phosphorylation of these proteins, while Grb2 knockdown only slightly increased transmembrane Ca2+ mobilization. Of note, concomitant HGAL expression and Grb2 knockdown further increased intracellular and transmembrane Ca2+ influx and phosphorylation of BCR effectors in comparison to HGAL expression alone. Expression of the HGAL (FFENV) mutant also enhanced Ca2+ influx and phosphorylation of BCR effectors in comparison to wild type HGAL. Concordantly, expression of the dominant negative Grb2 (W193K) mutant also enhanced HGAL's effects on BCR signaling. These observations suggest that Grb2's interaction with HGAL ameliorates HGAL's effects on BCR signaling.We previously showed that HGAL interacts with Syk and enhances Syk kinase activity. We now demonstrate that Grb2 Co-IPs with both Syk and HGAL and thus may potentially interfere with HGAL-Syk interaction. Indeed, knockdown of Grb2 increased HGAL Co-IP with the Syk kinase and this was associated with increased BCR signaling. These findings indicate that Grb2 ameliorates HGAL-mediated enhancement of BCR signaling by decreasing HGAL binding to Syk.In summary, out data demonstrates that Grb2 directly interacts with HGAL and ameliorates HGAL-enhanced BCR signaling. These interactions may play an important function in regulating the magnitude of BCR signaling during the GC reaction. DisclosuresNo relevant conflicts of interest to declare.

Usefulness of HGAL and LMO2 Immunohistochemistry in the Identification of Follicular Lymphomas of the Non-Gastric Gastrointestinal Tract

We studied the sensitivity of 2 relatively new markers of germinal center B-cell origin, namely human germinal center-associated lymphoma (HGAL) and Lim-only transcription factor 2 (LMO2), in the identification of follicular lymphomas (FLs) of the nongastric gastrointestinal (GI) tract. We retrospectively reviewed cases of endoscopically derived primary, nongastric GI lymphomas including FL, grade 1 or 2, and extranodal marginal zone lymphoma (ENMZL) of mucosa-associated lymphoid tissue, classified based on morphologic features and immunohistochemical analysis. HGAL and LMO2 immunohistochemical stains were then prospectively performed in each case. When discrepant immunohistochemical results were obtained, fluorescent in situ hybridization was performed for t(14;18) IGH/BCL2 and IGH rearrangement using a dual color fusion and a dual color break-apart probe, respectively. All but one of the CD10-negative ENMZL cases were negative for both HGAL and LMO2. One case originally classified as ENMZL was positive for both HGAL and LMO2. Fluorescent in situ hybridization did not detect either t(14;18) IGH/BCL2 or IGH rearrangement in this case. It is likely, based on positivity of 2 established germinal center B-cell markers, that this represents a FL which was originally misclassified as an ENMZL based on CD10 negativity. Of the cases of FL (all CD10 and/or BCL-6 positive), 8 (80%) were positive for both HGAL and LMO2. Although HGAL and LMO2 did not demonstrate an increased sensitivity in the identification of FL of the nongastric GI tract in this series, they still were helpful in the reclassification of one of our cases, and may therefore be useful adjuncts in the identification of FL of the nongastric GI tract.

Interaction of HGAL with Grb2 Enhances RhoA and B-Cell Receptor Signaling

AbstractAbstract 681Expression of the Human Germinal center Associated Lymphoma (HGAL) gene is restricted to germinal center (GC) B-lymphocytes and GC-derived lymphomas. HGAL expression identifies lymphomas characterized by improved survival. Previously we have demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by 1) interaction with F-actin and myosin II, inhibiting the ability of myosin to translocate actin; 2) activating RhoA signaling by direct interactions with RhoA-specific guanine nucleotide exchange factors. We recently demonstrated that HGAL enhances B-cell receptor (BCR)-mediated Syk activation by directly binding to and enhancing Syk kinase activity. However, the precise molecular mechanisms of the HGAL effects on these signaling pathways and requirement for cofactors have yet to be described. Examination of the HGAL protein sequence revealed the presence of a putative growth factor receptor-bound protein 2 (Grb2) binding motif (YYENV; aa106-110). Grb2 is an abundant adaptor protein with important functions in multiple intracellular pathways, including BCR and RhoA signaling. We examined whether Grb2 may directly interact with the HGAL protein and be involved in regulation of HGAL-mediated signaling. Confocal microscopy studies demonstrated colocalization of HGAL and Grb2 proteins in Raji and HeLa cell lines, the latter transiently expressing HGAL protein. Concordantly, coimmunoprecipitation experiments in VAL and Raji lymphoma cell lines detected endogenous Grb2 in immunoprecipitates of endogenous HGAL and vice versa. GST pull down assay using recombinant GST-Grb2 failed to pull down non-phosphorylated TRX-HGAL protein. In contrast, TRX-HGAL protein in vitro phosphorylated by Lyn kinase was detected in the GST-Grb2 pull down, confirming a direct interaction between these proteins and demonstrating requirement for HGAL phosphorylation, as would be predicted from the YYENV binding motif. We further elucidated the relevance of this interaction by evaluating the effects of siRNA mediated knockdown of Grb2, HGAL or both proteins together on BCR and RhoA signaling in lymphoma cell lines. In accordance with our previous data, HGAL knockdown led to a significant decrease in phosphorylated Syk (Y352) following BCR stimulation. A similar decrease in Syk(P352) phosphorylation was observed following knockdown of Grb2 alone or simultaneously with HGAL. Concordantly, BCR-induced Ca2+ mobilization was decreased significantly and to a similar extent following HGAL or Grb2 knockdown. Concomitant knockdown of HGAL and Grb2 did not lead to additive or synergistic decrease in BCR-induced Ca2+ mobilization. Similarly, knockdown of HGAL or Grb2 decreased fibronectin(FN)-induced RhoA-GTP levels. To further elucidate the importance of the HGAL-Grb2 interaction, we have generated a plasmid encoding a HGAL(Y106FY107F) protein in which the putative Grb2 binding site was mutated. The HGAL(Y106FY107F) protein is not interacting with the Grb2 protein, as demonstrated by coimmunoprecipitation studies. Expression of the wild type HGAL protein in HeLa cells markedly increased both FN-induced RhoA-GTP levels and RhoA-mediated serum response factor (SRF)-induced transcriptional activity from the serum responsive element (SRE). In contrast, the HGAL(Y106FY107F) mutant did not affect either FN-induced RhoA-GTP levels or SRF-induced transcriptional activity from the SRE. These observations suggest that the HGAL-Grb2 interaction is important for mediating HGAL's effect on RhoA signaling pathway. HGAL was previously shown to inhibit lymphoma cell motility by activating the RhoA signaling pathway. Indeed, siRNA mediated HGAL knockdown significantly increased lymphoma cell chemotaxis in response to IL-6 and SDF1. Knockdown of Grb2 similarly increased IL-6-induced lymphoma chemotaxis, with no further increases in IL-6 induced chemotaxis observed flowing concomitant HGAL and Grb2 knockdown. In contrast, compared to the HGAL knockdown, knockdown of Grb2 significantly increased lymphoma cell chemotaxis in response to SDF-1. Concomitant knockdown of both Grb2 and HGAL resulted in an ever greater increase in lymphoma cell motility in response to SDF-1. In summary, our findings suggest that interaction of HGAL with Grb2 protein enhances both RhoA and B-cell receptor signaling and may be necessary for mediating at least some of the HGAL effects. Disclosures:No relevant conflicts of interest to declare.

HGAL-a Germinal Center Specific Protein, Enhances B-Cell Receptor Signaling by Activation of Syk, Leading to Follicular Lymphoproliferation

Abstract 584The Human Germinal center Associated Lymphoma (HGAL) gene is exclusively expressed in germinal center (GC) B-lymphocytes and GC-derived lymphomas. In patients with diffuse large B-cell lymphomas (DLBCL), HGAL expression identifies a subgroup of patients with biologically distinct tumors associated with improved survival. Our previous in vitro studies demonstrated that HGAL decreases spontaneous and chemoattractant-induced cell motility by activating the RhoA signaling pathway and by directly interacting and augmenting F-actin and myosin II binding. However, the major function of HGAL in GC lymphocytes remains largely unknown. Based on our previous observation of tyrosine phosphorylation of a modified ITAM motif in the HGAL by Lyn, we hypothesized that HGAL may be involved in B-cell receptor (BCR) signaling. Indeed, following BCR stimulation of two GCB-like lymphoma cell lines (Raji and VAL), we observed marked reduction of Syk, Btk and PLCγ phosphorylation upon knockdown of endogenous HGAL by specific but not control siRNAs. Concordantly, HGAL knockdown in BCR-stimulated Raji cells reduced Ca2+ mobilization and decreased NFAT transcriptional activity as analyzed by a luciferase reporter assay. HGAL expression in the BCR-stimulated HBL1 lymphoma cell line (lacking endogenous HGAL protein) resulted in increased Syk, Btk and PLCγ phosphorylation. Syk plays a major role in coupling BCR activation to downstream effectors. Endogenous HGAL was detected in immunoprecipitates of endogenous Syk and vice versa. Nanoscope microscopy studies confirmed co-localization of HGAL and Syk proteins in cell membranes, which was enhanced following BCR stimulation. In BCR-stimulated cells, Syk kinase activity was markedly increased following addition of HGAL protein as measured by an in vitro Syk kinase activity assay. To comprehensively examine HGAL effects on immune system and BCR signaling, we generated a transgenic mouse model in which HGAL is expressed under the control of the mouse Ly-6E.1 promoter in Sca1+ hematopoietic stem cells and progenitors of C57BL/6 × CBA mice. The Sca1-HGAL transgenic mice showed normal embryonic and post natal development, and at 8 weeks of age demonstrated normal lymphoid development without any significant changes in the major hematopoietic compartments (bone marrow (BM), spleen, thymus and peripheral lymph nodes) and in peripheral blood. They also exhibited normal GC development in response to a T-cell dependent antigen immunization. In contrast, at 12 months of age the Sca1-HGAL mice developed a decrease in BM immature B-cells at the expense of recirculating B-cells (B220+IgDhi) compared to the age-matched normal littermates, suggesting a defect in B-cell lymphopoiesis. All the Sca1-HGAL transgenic mice became ill from approximately 12 months of age and all died between 12 to 22 months of age with statistically shorter survival as compared to the wild type controls. Analysis of these animals showed massive splenomegaly with marked white pulp hyperplasia and presence of multiple, frequently contiguous nodules predominantly composed of polyclonal follicular (B220+CD21intCD23hi) B lymphocytes. Extra-lymphatic infiltration by similar B lymphocytes was observed in the liver, lungs and kidneys of Sca1-HGAL mice with advanced disease. IgG isotype titers in these animals tended to be higher than in the wild-type controls, reaching a statistically significant difference for the IgG1 isotype. Follicular hyperplasia in the Sca1-HGAL transgenic mice is likely attributable to increased RhoA activation and enhanced BCR signalling manifested by increased Syk phosphorylation, Ca2+ mobilization and in vitro B cell proliferation following BCR stimulation, in agreement with similar data observed in human DLBCL cell lines expressing HGAL. Gene expression profiling of lymphoid tissues confirmed significantly enhanced BCR signalling and RhoA pathway activation in Sca1-HGAL transgenic mice, corresponding to similar pathway activation in human lymphoma cell lines over-expressing HGAL. Overall, our findings demonstrate that HGAL, specifically expressed in GC B cells, enhances responsiveness to antigens by stimulating Syk kinase activity that without appropriate regulation may lead to lymphoproliferation. Further studies are needed to examine the role of HGAL in the pathogenesis of GC-derived lymphomas. Disclosures:No relevant conflicts of interest to declare.

Comparison of germinal center markers CD10, BCL6 and human germinal center-associated lymphoma (HGAL) in follicular lymphomas

BackgroundRecently, human germinal center-associated lymphoma (HGAL) gene protein has been proposed as an adjunctive follicular marker to CD10 and BCL6.MethodsOur aim was to evaluate immunoreactivity for HGAL in 82 cases of follicular lymphomas (FLs) - 67 nodal, 5 cutaneous and 10 transformed - which were all analysed histologically, by immunohistochemistry and PCR.ResultsImmunostaining for HGAL was more frequently positive (97.6%) than that for BCL6 (92.7%) and CD10 (90.2%) in FLs; the cases negative for bcl6 and/or for CD10 were all positive for HGAL, whereas the two cases negative for HGAL were positive with BCL6; no difference in HGAL immunostaining was found among different malignant subtypes or grades.ConclusionsTherefore, HGAL can be used in the immunostaining of FLs as the most sensitive germinal center (GC)-marker; when applied alone, it would half the immunostaining costs, reserving the use of the other two markers only to HGAL-negative cases.

PRDM1/Blimp1 downregulates expression of germinal center genes LMO2 and HGAL

Human germinal center-associated lymphoma (HGAL) and LIM domain only-2 (LMO2) are proteins highly expressed in germinal center (GC) B lymphocytes. HGAL and LMO2 are also expressed in GC-derived lymphomas and distinguish biologically distinct subgroups of diffuse large B-cell lymphomas (DLBCL) associated with improved survival. However, little is known about their regulation. PRDM1/Blimp1 is a master regulator of terminal B cell differentiation and may also function as a tumor suppressor in the pathogenesis of DLBCL, where it is frequently inactivated by mutations and deletions. We now demonstrate that both HGAL and LMO2 are directly regulated by the transcription repressor PRDM1. In vivo studies demonstrate that PRDM1 directly binds to the recognition sites within the upstream promoters of both HGAL and LMO2. PRDM1 binding suppresses endogenous protein and mRNA levels of HGAL and LMO2. In addition, promoter analysis reveals that site-specific binding of PRDM1 to the promoters is capable of repressing transcriptional activity. This inhibitory effect of PRDM1 suggests that it has a key role in the loss of HGAL and LMO2 expression upon differentiation of GC B cells to plasma cells and may also contribute to absence of HGAL and LMO2 expression in post-GC lymphoid tumors.

HGAL Protein Expression Persists in Disorders of Germinal Center Dissolution

Human Germinal Center-associated Lymphoma (HGAL) is a germinal center (GC) B-cell marker associated with a favorable outcome in diffuse large B-cell and classic Hodgkin lymphomas (CHL). To test its potential role in GC function, 75 cases involving GC disruption including 23 progressive transformation of germinal centers (PTGC), 25 follicle lysis and 27 Castleman disease (CD) were studied. HGAL protein expression uniformly correlated with GC B-cells in all except a subset of hyaline-vascular CD that showed severe regression of GCs. HGAL staining highlighted dismantled GCs in PTGC, in contrast to weak or absent CD10 and BCL6 staining. In follicle lysis, HGAL staining was comparable to that of CD10, BCL6, and CD21 in highlighting lysed follicles. Our findings show that HGAL protein expression effectively discriminates clusters of GC B-cells in disrupted follicles. Its persistence in disrupted GCs, suggests that it may be necessary for GC maintenance and supports its proposed role of confining B-cells to the GC microenvironment.

Germinal center-specific protein human germinal center associated lymphoma directly interacts with both myosin and actin and increases the binding of myosin to actin

Human germinal center associated lymphoma (HGAL) is a germinal center-specific gene whose expression correlates with a favorable prognosis in patients with diffuse large B-cell and classic Hodgkin lymphomas. HGAL is involved in negative regulation of lymphocyte motility. The movement of lymphocytes is directly driven by actin polymerization and actin-myosin interactions. We demonstrate that HGAL interacts directly and independently with both actin and myosin and delineate the HGAL and myosin domains responsible for the interaction. Furthermore, we show that HGAL increases the binding of myosin to F-actin and inhibits the ability of myosin to translocate actin by reducing the maximal velocity of myosin head/actin movement. No effects of HGAL on actomyosin ATPase activity and the rate of actin polymerization from G-actin to F-actin were observed. These findings reveal a new mechanism underlying the inhibitory effects of germinal center-specific HGAL protein on lymphocyte and lymphoma cell motility.

Immunoarchitectural Patterns in Follicular Lymphoma: Efficacy of HGAL and LMO2 in the Detection of the Interfollicular and Diffuse Components

Follicular lymphoma (FL) can exhibit variant histologic patterns that can lead to confusion with other B-cell lymphomas and reactive conditions. Diagnostic markers such as CD10 and BCL2 may be difficult to interpret in variant FL patterns, and are often diminished or absent in the interfollicular and diffuse components. We evaluated 2 recently characterized germinal center B-cell markers, human germinal center associated lymphoma (HGAL), and LIM-only transcription factor 2 (LMO2), in 127 FL patient biopsies (94 nodal, 33 extranodal), and correlated the findings with histologic pattern, cellular composition, grade, and additional immunostains (CD20, CD3, CD21, CD10, BCL2, and BCL6). Architectural patterns included predominantly follicular (75%) and follicular and diffuse components (25%); 10 cases showed marginal zone differentiation and 3 were floral variants. Eighty-nine cases were low grade (38 grade 1; 51 grade 2) and 38 were grade 3 (29 grade 3A and 9 grade 3B). HGAL had the highest overall sensitivity of detecting FL and was superior in detecting the interfollicular and diffuse components compared with BCL2, LMO2, CD10, and BCL6. All 28 cases that lacked CD10, expressed HGAL, and the majority also expressed LMO2. Our results show that HGAL and LMO2 are sensitive markers for FL diagnosis. The addition of HGAL and LMO2 to the immunohistologic panel is beneficial in the work-up of nodal and extranodal B-cell lymphomas and the efficacy of HGAL in detecting the follicular, interfollicular and diffuse components of FL is of particular value in the setting of variant immunoarchitectural patterns.

Human Germinal Center-Associated Lymphoma (HGAL) Protein Expression Is Associated with Improved Failure-Free Survival in Brazilian Patients with Classical Hodgkin Lymphoma.

Abstract 4632 BACKGROUNDThe HGAL gene has prognostic value in diffuse large B-cell lymphoma and expression of its cognate protein is germinal center–specific. A previous study had suggested that HGAL protein expression might also be related to outcome in patients with Hodgkin lymphoma. PATIENTS AND METHODSThe aim of the current study was to confirm the prognostic impact of HGAL protein expression in an independent, well-characterized cohort of 232 classic Hodgkin lymphoma patients treated uniformly with the ABVD regimen from 1997 to 2004 in Brazil. The median follow-up was 6.2 years. RESULTSTissue microarray analysis showed HGAL staining in 188 specimens (81%). The overall survival was better in patients with young age, early-stage, absence of B symptoms, low-risk international prognostic score (IPS) and good performance status. Failure-free survival was superior in patients with early-stage disease, low-risk IPS and HGAL-positive patients. The estimated 5-year failure-free survival for HGAL-positive and HGAL-negative patients was 82% and 67%, respectively (P=0.03). When stage, B symptoms and performance status were included along with HGAL in a multivariate analysis, advanced stage and absence of HGAL staining were independent predictors of a worse failure-free survival. CONCLUSIONThis study confirms and validates recent findings of a correlation between HGAL expression and outcome in classical Hodgkin lymphoma. Disclosures:No relevant conflicts of interest to declare.

Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma

The human germinal center-associated lymphoma (HGAL) gene has prognostic value in diffuse large B-cell lymphoma, and expression of its cognate protein is germinal center-specific. A previous study had suggested that HGAL protein expression might also be related to the outcome in patients with Hodgkin lymphoma (HL). The aim of this study was to confirm the prognostic impact of HGAL protein expression in an independent, well-characterized cohort of 232 patients with classic HL treated uniformly with doxorubicin, bleomycin, vinblastine and dacarbazine (ABVD). Tissue microarray analysis showed HGAL staining in 188 specimens (81%). Failure-free survival (FFS) was superior in patients with early-stage disease, low-risk IPS, and HGAL-positive patients. The estimated 5-year FFS for HGAL-positive and HGAL-negative patients was 82% and 67%, respectively (p = 0.03). In the multivariate analysis, advanced stage and absence of HGAL staining were independent predictors of a worse FFS. This study confirms and validates recent findings of a correlation between HGAL expression and outcome in classical HL.

Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma

Human germinal center-associated lymphoma protein expression is associated with improved failure-free survival in Brazilian patients with classical Hodgkin lymphoma

Expression of HGAL in primary cutaneous large B-cell lymphomas: evidence for germinal center derivation of primary cutaneous follicular lymphoma

The classification of primary cutaneous large B-cell lymphoma (PCLBCL) is based on standard morphology, immunohistochemistry, and clinical presentation. There are two major subtypes in the current WHO–EORTC classification: follicle center lymphoma and diffuse large B-cell lymphoma, leg-type (DLBCL-LT). The goals of this study were to examine a series of DLBCLs to determine (1) whether the immunohistochemical paradigm of germinal center B-cell and non-germinal center B-cell types of systemic DLBCL could be applied to PCLBCL; (2) whether application of the newly described germinal center B-cell marker, human germinal center-associated lymphoma (HGAL) also discriminates between these types as a further support for germinal center B-cell origin for primary cutaneous center lymphoma; and (3) whether any of these biologic markers were of prognostic significance. To this end, 32 cases of diffuse PCLBCL (22 primary cutaneous follicular center lymphomas and 10 DLBCL-LT) were classified based on the WHO–EORTC criteria and studied for expression of CD20, BCL2, BCL6, CD10, MUM-1, and HGAL by immunohistochemistry. Results were correlated with clinical features. HGAL and BCL6 expression and germinal center B-cell phenotype were associated with primary cutaneous follicular center lymphoma. The combination of HGAL and BCL6 positivity had the highest sensitivity (88%) and specificity (100%) for predicting subtype compared to either marker alone. Both HGAL and BCL6 were associated with the germinal center B-cell phenotype. The correlation of HGAL expression with the germinal center B-cell phenotype demonstrates the role of this marker in the classification of cutaneous large B-cell lymphomas. BCL6 expression was the only immunohistochemical marker associated with overall survival. Characterizing PCLBCLs with markers of B-cell maturation stage is a useful framework for studying, classifying, and clinically stratifying these lymphomas.