non-Hodgkin Lymphoma Specimens Research Articles

Immunohistochemical study of microvessel density in Non-Hodgkin lymphoma

Background Non-Hodgkin lymphoma (NHL) is considered one of the most common malignancies in Egypt. Angiogenesis plays a role in the biology of lymphomas. Several methods are used to assess angiogenesis, of which microvessel density (MVD) is considered to be the most representative one. CD31 is a marker of vascular endothelial cells used for assessment of MVD. The study of MVD in NHL has not been fully tested especially in our locality. Aim To evaluate MVD in different subtypes of NHL and reactive hyperplasia (RH) by immunohistochemical staining using anti-CD31. To correlate the expression of CD31 in NHL with the available clinicopathological features. Materials and methods Immunohistochemical study was done on 52 specimens of NHL (24 diffuse large B-cell lymphoma, 11 follicular lymphoma, 11 B-small lymphocytic lymphoma, and six T-cell lymphoma) and 10 specimens of RH of lymph node (control group). Results CD31 expression was significantly higher in NHL than RH. Level of CD31 expression varied significantly between different subtypes of NHL. Follicular lymphoma showed the highest expression, whereas B-small lymphocytic lymphoma showed the lowest. A weak positive significant correlation between CD31 and Ki67 expression was found in NHL specimens. Insignificant difference was detected between CD31 expression and all studied clinical features. Conclusion Angiogenesis is an essential process in the pathogenesis of NHL and an important biological factor that affects heterogeneity and aggressiveness of NHL.

Expression and Function of Tigit in B-Cell Non-Hodgkin Lymphoma

Background:T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and a novel member of CD28 family. TIGIT is expressed on NK cells, effector and memory T cells, and Treg cells. Upon ligation with CD155, TIGIT delivers an inhibitory signal and negatively regulates anti-tumor responses. While important in normal T-cell biology, the expression and function of TIGIT in the blood and tumor microenvironment of patients with B-cell non-Hodgkin lymphoma (NHL) is completely unknown. Goal: To phenotypically and functionally characterize TIGIT+T cell subsets in B-cell NHL and compare expression of TIGIT on intratumoral T cells to normal controls. Results: In peripheral blood, TIGIT expression was not detected on resting T cells. In contrast, when we analyzed biopsy specimens from B-cell NHL patients, we observed that TIGIT is variably but highly expressed on a subset of intratumoral T cells. A median of 41.1% (range: 24.9-56.1, n=5) of CD4+ or 22.2% (range: 13.4-33.3, n=5) of CD8+ T cells from tumor specimens expressed TIGIT on the cell surface. In normal tonsil tissue, expression level of TIGIT was modest or negligible. A median of 12.0% (range: 9.1-15.8, n=5) of CD4+ or 3.9% (range: 1.3-5.7, n=5) of CD8+ T cells express TIGIT on the cell surface, which is significantly lower than that in B-cell NHL (p=0.005 and p=0.003 for CD4+ and CD8+, respectively). We observed that both CD45RA+ and CD45RA- T cells express TIGIT with the greatest expression on the CD45RA- population in B-cell NHL specimens. Furthermore, we found that TIGIT+ T cells coexpressed other immune checkpoint molecules including PD-1 and TIM-3. Functionally, TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and IL-2-producing cells was lower in the TIGIT+ population than in TIGIT-T cells. Unlike TIM-3 that is coexpressed with PD-1 and whose expression is upregulated by IL-12, TIGIT expression is not upregulated by IL-12, suggesting that a different mechanism is involved in TIGIT upregulation in B-cell NHL. Conclusion: Taken together, these results indicated that TIGIT is highly expressed on intratumoral T cells in B-cell NHL and is expressed on a population of T-cells with suppressed immune function. TIGIT, while co-expressed with PD-1 and TIM-3, is not upregulated by cytokines such as IL-12, suggesting that it is regulated by an alternate pathway. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression and exhaustion in B-cell NHL. Disclosures No relevant conflicts of interest to declare.

Signal-Regulatory Protein-α (SIRP- α) Expression Delineates Distinct Subsets in Monocytes/Macrophages in Normal Tissue and in B-Cell Non-Hodgkin Lymphoma

BACKGROUNDMonocytes and macrophages (mo/mΦ) are a key part of the composition of peripheral blood (PB) and tissues and increased numbers of mo/mΦ have been associated with patient outcome in non-Hodgkin lymphoma (NHL). Because CD14 is abundantly expressed on the surface of human mo/mΦ, it is often used to identify or isolate human mo/mΦ, and immunosuppressive CD14+HLA-DRlow monocytes have been shown to be increased in the peripheral blood of NHL patients. However, we have previously shown that CD14 expression on mo/mΦ is substantially lower than CD68 expression suggesting that many CD68+ mo/mΦ are CD14 negative, especially in spleen and lymph node tissues. To characterize both CD14+ and CD14- mo/mΦ in PB and tissues, we isolated all mo/mΦ from B-cell NHL specimens and normal controls and assessed their phenotype and function.METHODSHuman mo/mΦ were isolated by negative selection from PB and tissue biopsy specimens (B-cell NHL and normal tissues) using the immunomagnetic isolation (monocyte enrichment kit). Morphological and immunophenotypic characteristics of isolated mo/mΦ were determined by Giemsa stain and flow cytometry. Phagocytosis and migration assays were used to determine the function of isolated mo/mΦ. T cells were co-cultured with mo/mΦ and T cell proliferation was evaluated by CFSE staining and detected by flow cytometry.RESULTSUsing a monocyte enrichment kit to isolate all mo/mΦ, the purity of isolated mo/mϕ was 85~99%, which was defined by the percentage of lineage-negative cells (i.e. cells without expression of CD3,CD19, CD20, and CD56). We found that these isolated mo/mΦ constituted 2 populations: a more frequent population of larger cells and a less common population of smaller cells. In contrast to PB, CD14 positive mo/mΦ constituted less than 40% of the tissue mo/mΦ from the isolated population. Furthermore, we found that the cell size from CD14+ mo/mΦ were larger than CD14- mo/mΦ. Using CD14 and SIRP-α, we could identify 3 populations of mo/mΦ: CD14+SIRP-αhigh, CD14-SIRP-αdim and CD14-SIRP-α- cells. CD14+SIRP-αhigh cells and CD14-SIRP-αdim cells typically constituted the population of larger cells, while CD14-SIRP-α- cells constituted the population of smaller cells. CD14-SIRP-α- cells lacked the typical phenotypic markers and had decreased phagocytic and migratory ability compared to CD14+SIRP-αhigh and CD14-SIRP-αdim cells. Furthermore, we found that these 3 populations of mo/mΦ had a differential effect on activated T-cells and that the CD14-SIRP-α- cells appeared increased number in biopsy specimens from NHL when compared to normal tissues.CONCLUSIONSWe have identified a unique population of small CD68+ mo/mΦ that lack expression of CD14, SIRP-α, and other FcγR markers. This subset of mo/mΦ is more prevalent in NHL tissues and has limited phagocytic and migratory functions. This CD14-SIRP-α- mo/mΦ subpopulation may play an inhibitory role in anti-cancer and inflammatory responses. DisclosuresNo relevant conflicts of interest to declare.

Targetable subsets of non-Hodgkin lymphoma in Malawi define therapeutic opportunities

AbstractDiagnostics and supportive care for patients with non-Hodgkin lymphoma (NHL) in lower- and middle-income countries (LMICs) are lacking. We hypothesized that high-throughput transcription-based diagnostics could classify NHL specimens from Malawi amenable to targeted therapeutics. We established tissue microarrays and classified 328 cases diagnosed by hematoxylin and eosin as NHL at University of Malawi College of Medicine using immunohistochemistry (IHC) for conventional markers and therapeutic targets. A subset was analyzed using NanoString-based expression profiling with parsimonious transcriptional classifiers. Overall, 72% of lymphomas were high-grade B-cell tumors, subsets of which were enriched for expression of MYC, BCL2, and/or PD-L1. A 21-gene transcriptional classifier, previously validated in Western cohorts, divided 96% of diffuse large B-cell lymphomas (DLBCLs) with 100% of B-cell lymphomas, unclassifiable, into 1 cluster and 88% of Burkitt lymphomas into a separate cluster. Cell-of-origin categorization of 36 DLBCLs by NanoString lymphoma subtyping test (LST) revealed 69% concordance with IHC. All discordant cases were classified as germinal center B cell–like (GCB) by LST but non-GCB by IHC. In summary, utilization of advanced diagnostics facilitates objective assessment and segregation of biologically defined subsets of NHL from an LMIC without expert review, thereby establishing a basis for the implementation of effective and less toxic targeted agents.

CD37 Is a Potential Therapeutic Target for B-Cell Non-Hodgkin Lymphoma

AbstractAbstract 3098 Background:The Non-Hodgkin Lymphomas (NHLs) are a heterogeneous group of malignancies with approximately 85% of NHL belonging to the B-cell lineage. The use of monoclonal antibodies (mAb), including the anti-CD20 therapy rituximab, has become a standard of care for the treatment of many forms of NHL. However, only a subset of patients respond to rituximab and the majority of those eventually relapse after treatment. Additional therapeutic options are necessary for these patients. Here we report the expression of CD37, a lineage-specific B-cell antigen, in different subtypes of B-cell NHL tissues and evaluate the functional activity of an anti-CD37 scFv-Fc fusion protein, known as a SMIP ™ protein (CD37-SMIP) against NHL cells. Methods:Fresh NHL tissues or peripheral blood from leukemia-stage lymphoma patients were used for flow cytometric assay of CD37 expression. Monotypic kappa or lambda IgG light chain positive malignant B-cells were gated for this assay. Immunohistochemical (IHC) staining of formalin fixed paraffin-embedded (FFPE) NHL tissues was performed for CD37 expression. Fresh primary NHL cells were isolated and used as targets in assays to measure antibody dependent cellular cytotoxicity (ADCC) activity as well as direct cell death mediated by CD37-SMIP. The effects of CD37-SMIP on multiple B-cell NHL lines was also determined by measuring cell proliferation, Annexin V staining, caspase3/7 release and changes in mitochondria membrane potentials using JC-1 staining. Results:Eighteen cases of NHL (follicular lymphoma (2), chronic lymphocytic leukemia/small lymphocytic lymphoma (3), mantle cell lymphoma (5), persistent marginal zone lymphoma (1), diffuse large B-cell lymphoma (1) and other types (6)) were analyzed by flow cytometry. Six of the eighteen cases had a history of rituximab treatment. Flow data showed malignant B-cells from these NHL specimens were strongly positive for CD37, while other cell populations had minimal to low levels of CD37 expression. IHC results on FFPE samples (n=170) indicated 89% (151 of 170 cases) of stained lymphoma tissues were positive for CD37, including follicular lymphoma (80/88), mantle cell lymphoma (39/44), DLBCL (32/38), and marginal zone lymphoma (5/5). Twenty seven B-cell lymphoma cell lines representing MCL, DLBCL, FL, and Burkitt's lymphoma subtypes were evaluated for CD37 expression and functional response to CD37-SMIP. CD37-SMIP mediated cell death in a wide range of NHL cell lines. The response to CD37-SMIP correlated with the levels of CD37 surface expression in NHL cell lines (Pearson analysis, r=0.78) The CD37-SMIP mediated response was rapid, with Annexin V staining and mitochondria depolarization observed in 3 hours; however, no caspase activity was observed at this time point. Two of ten NHL cell lines had modest caspase 3/7 activity at 24 h post treatment. However, caspase inhibitors did not suppress the CD37-SMIP mediated response. ADCC and direct killing activities elicited by SMIP-CD37 on freshly isolated primary NHL cells are under evaluation. Conclusion:CD37 is highly expressed on majority of B-cell NHL tissues, including those from patients treated with rituximab. CD37-SMIP mediated rapid cell death in NHL cell lines with mitochondria dysfunction but appears to be independent of caspase activity. The expression pattern of CD37 and functional effects of CD37-SMIP supports the targeting of CD37 in B-cell NHL. TRU-016, a humanized SMIP protein specific for CD37, is currently being evaluated in relapsed CLL and NHL patients in a phase 1 trial. Disclosures:No relevant conflicts of interest to declare.

Serological Identification of HSP105 as a Novel Non-Hodgkin Lymphoma Therapeutic Target

AbstractAbstract 463We recently reported that vaccination with autologous monocyte-derived dendritic cells pulsed with dying autologous tumor cells elicited a clinical response strongly associated with multifaceted antitumor immune-activation in relapsed indolent non-Hodgkin lymphoma (NHL) patients. We have now set out to determine whether vaccine-induced humoral response is directed against common indolent NHL-restricted antigens (ags), which could thus be exploited as novel targets for therapy. Antibodies (Abs) were purified from pre- and post-vaccine patients' serum samples, biotin-conjugated, and initially tested by immunohistochemistry (IHC) and flow cytometry (FC) on allogeneic tumors biopsies or live tumor cells, both primary tumors and cell lines. We found that post-vaccine Abs from responders (R) reacted with allogeneic NHL at significantly higher levels than their matched pre-vaccine samples or non-R Abs. Furthermore, Rs' post-vaccine sera significantly impaired the growth of DOHH-2 and RL-19 follicular lymphoma (FL) cell lines, as revealed by standard 3-(4,5- dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. To identify the therapeutically targeted NHL ags, we then used biotin-conjugated patient Abs to immunoblot one-dimensional SDS-PAGE of DOHH-2 cell protein fractions obtained by isoelectrofocusing. Protein bands differentially revealed by post-vaccine Abs from R were analyzed by Mass Spectrometry (MS). One differential band migrating at about 100 kDa was revealed among the most acidic protein fractions only when post-vaccine samples from R were used. MS analysis identified heat shock protein (HSP) 105 in the differentially reacting bands. Immunoprecipitation with a commercial anti-HSP105 Ab followed by Western blot analysis with biotin-conjugated pre- and post-vaccine Abs from R confirmed the increased ability of the post-vaccine sample to recognize HSP105. FC disclosed HSP105 both on the cell membrane and in the cytoplasm of a panel of B-cell NHL cell lines and normal B cells. The extent of HSP105 surface expression increased in function of lymphoma istotype aggressiveness. The antitumor activity of anti-HSP105 Ab, measured by MTT assays, was thus higher against Burkitt's lymphoma (BL) cell lines than germinal centre-derived diffuse large B cell lymphoma and FL cell lines, which displayed an IC50 of 4.5, 7.5, 11.7 μg/ml, respectively. To confirm these finding in primary human tumors, we performed IHC analyses of HSP105 on 68 diagnostic NHL specimens (35 low-grade and 33 high-grade NHL) and 23 non-malignant lymph nodes obtained from our Institutional Tissue Bank. Low-grade NHL's cytoplasmic immunoreactivity was mainly restricted to actively proliferating cells (i.e. Ki67 positive germinal centre cells), whereas high-grade NHL displayed a significantly higher expression of HSP105, measured both as intensity and percentage of positive cells (p=0.0002). In addition, malignant cells in high-grade NHL more often displayed a specific cell-surface staining. Interestingly, the expression pattern and intensity of HSP105 was widely superimposable on that of the proliferation marker Ki67, as detected by the specific monoclonal Ab Mib-1. Lastly, the therapeutic effects of HSP105 functional inhibition was studied in Namalwa BL xenotransplanted SCID mice using a specific commercial Ab. Treated mice showed a significant delay in tumor growth compared to untreated control animals (p=0.0014). Taken as a whole, our results indicate that HSP105 could be a new potential biotarget for the treatment of NHL and a novel candidate biomarker for an improved management of B-cell lymphoma. Its location on the normal B cell surface, and its increasing expression with NHL aggressiveness open a new area in which to assess its role in B-cell biology and lymphoma physiopathology. Disclosures:No relevant conflicts of interest to declare.

Serum Adiponectin As a Predictor of Childhood Non-Hodgkin's Lymphoma: A Nationwide Case-Control Study

To our knowledge, this is the first study exploring the association of childhood non-Hodgkin's lymphoma (NHL) with serum adiponectin and leptin levels in a nationwide case-control series. In addition, expression of adiponectin receptors in NHL specimens was assessed, and the association between adipokines and childhood NHL survival and prognosis was examined. We studied 121 incident childhood (0 to 14 years) NHL cases registered in the Nationwide Registry for Childhood Hematological Malignancies (1996 to 2006) and an equal number of matched controls, for whom sociodemographic, lifestyle, prenatal characteristics, and fasting blood serums were collected. Serum adiponectin and leptin levels were determined. Immunohistochemisty for adiponectin receptors expression was performed on commercially available adult NHL specimens (n = 30) and in a subset of childhood NHL cases (n = 6) that were available. Summary statistics, multiple conditional logistic regression analyses, and survival analysis were performed. Higher serum adiponectin, but not leptin, levels were independently associated with childhood NHL (odds ratio, 1.82; 95% CI, 1.30 to 2.56), after adjusting for obesity and established risk factors. Higher adiponectin levels at diagnosis were positively associated with relapse and poor survival, but hormone levels did not differ among NHL subtypes. Adiponectin receptors 1 and 2 were present in 90% and 57% of adult samples and in 83% and 100% of childhood NHL samples, respectively. Elevated serum adiponectin, but not leptin, levels are independently associated with childhood NHL and poor prognosis. Adiponectin receptors are expressed in NHL, suggesting that adiponectin may represent not only a potential clinically significant diagnostic and prognostic marker but also a molecule that may be implicated in NHL pathogenesis.

Malignant B Cell-Derived TGF-β Controls the Generation of TH1, TH17 and Treg Cells in the Tumor Microenvironment of B-Cell Non-Hodgkin Lymphoma (NHL).

Background: Our previous work has shown that malignant B cells induce the development of intratumoral Treg cells that inhibit the host anti-tumor response. In contrast to an increase in Treg cells, we found that the number of effector T helper cells (TH1, TH2 and TH17) was low in B-cell NHL tumors, suggesting an imbalance between Treg and TH cells in the tumor microenvironment. Understanding the mechanism(s) of this imbalance is important to the development of treatments to enhance host immunity and in previous work we have shown that signaling through CD70, CD80 and CD86 plays a role. Since soluble factors, particularly TGF-β, have an important role in directing T-cell differentiation, we evaluated in this study the role of TGF-β in the lymphoma microenvironment.Goal: To determine the effect of TGF-β on the generation of intratumoral TH1, TH17 and Treg cells in human B-cell NHL.Results: Human B-cell NHL specimens were obtained from consenting patients and were used for all experiments. Using an ELISA assay, we found that malignant B cells variably secrete TGF-β - median 100 pg/ml per million cells (range: undetectable −229 pg/ml, n=7). Using flow cytometry, we showed that addition of exogenous TGF-β enhanced the expression of Foxp3+ in activated CD4+ or CD4+CD45RA+ or CD4+CD45RO+ nodal T cells, suggesting that TGF-β promotes the generation of Treg cells in tumor microenvironment. In contrast, TGF-β suppressed expression of IFN-g in activated CD4+ T cells and inhibited the up-regulation of IL-12 and IL-23-induced IFN-γ expression in CD4+ cells, indicating that TGF-β suppresses the generation of TH1 cells. TGF-β alone slightly inhibited IL-17 expression in CD4+ T cells; however, TGF-β, in the presence of IL-6 and IL-23, upregulated IL-17 expression in CD4+ T cells, suggesting proinflammatory cytokines are able to reverse the suppression induced by TGF-β. These results indicate that TGF-β plays an important role in the regulation of intratumoral TH17 cell generation. In additional experiments, TGF-β was found to exert a suppressive effect on the proliferation of both CD4+ and CD8+ intratumoral T cells. However, treatment with TGF-β enhanced IL-2 production by intratumoral CD4+ T cells detected by intracellular staining of flow cytometry. Interruption of IL-2 signaling by anti-IL-2 Ab abolished the upregulation of TGF-β-mediated Foxp3 expression and enhanced the production of IL-17 in CD4+ T cells. Furthermore, treatment with anti-IL-2 Ab reversed the inhibition of NHL B cell-mediated TH17 cell generation.Conclusion: These results suggest that TGF-β controls the generation of TH1, TH17 and Treg cells contributing to the imbalance of effector TH cells and inhibitory Treg cells in the tumor microenvironment of B-cell NHL through IL-2. Since malignant B-cells produce TGF-β, these results further support the important role of malignant B cells in the regulation of intratumoral T cell generation and the host immune response.

Discovery of novel epigenetic markers in non-Hodgkin's lymphoma

Non-Hodgkin's lymphoma (NHL) is a group of malignancies with heterogeneous genetic and epigenetic alterations. Discovery of molecular markers that better define NHL should improve diagnosis, prognosis and understanding of the biology. We developed a CpG island DNA microarray for discovery of aberrant methylation targets in cancer, and now apply this method to examine NHL cell lines and primary tumors. This methylation profiling revealed differential patterns in six cell lines originating from different subtypes of NHL. We identified 30 hypermethylated genes in these cell lines and independently confirmed 10 of them. Methylation of 6 of these genes was then further examined in 75 primary NHL specimens composed of four subtypes representing different stages of maturation. Each gene (DLC-1, PCDHGB7, CYP27B1, EFNA5, CCND1 and RARbeta2) was frequently hypermethylated in these NHLs (87, 78, 61, 53, 40 and 38%, respectively), but not in benign follicular hyperplasia. Although some genes such as DLC-1 and PCDHGB7 were methylated in the vast majority of NHLs, others were differentially methylated in specific subtypes. The methylation of the candidate tumor suppressor gene DLC-1 was detected in a high proportion of primary tumor and plasma DNA samples by using quantitative methylation-specific PCR analysis. This promoter hypermethylation inversely correlated with DLC-1 gene expression in primary NHL samples. Thus, this CpG island microarray is a powerful discovery tool to identify novel methylated genes for further studies of their relevant molecular pathways in NHLs and identification of potential epigenetic biomarkers of disease.

The Humanized Anti-CD40 Antibody, SGN-40, Promotes Apoptosis Signaling and Is Effective in Combination with Standard Therapies in Lymphoma Xenograft Models.

CD40 is a TNF receptor family member that is expressed on B cells and by a wide variety of transformed cells including non-Hodgkin's lymphoma (NHL), multiple myeloma, and various solid tumors. The humanized anti-CD40 antibody, SGN-40, is a partial agonist that induces apoptosis as well as mediates ADCC against CD40+ NHL B cell lines, contributing to in vivo antitumor activity observed in human lymphoma xenograft models. The current study demonstrates the ability of SGN-40 to initiate multiple signaling cascades upon ligation of CD40 on NHL cell lines. SGN-40 was shown to activate the stress-induced p38 MAP kinase and pro-survival pathways including NF-κB, p42/44 MAP kinase and, to a lesser extent, AKT. Consistent with the apoptosis-inducing activity of SGN-40, cleavage of caspase-3 and its downstream substrate poly (ADP-ribose) polymerase was detected in NHL cell lines. SGN-40 signaling was qualitatively similar to that mediated by trimeric recombinant human CD40 ligand (rhCD40L). However, the overall magnitude of signaling was lower with SGN-40 compared to rhCD40L, consistent with the partial agonistic properties of SGN-40. In addition, constitutive phospho-AKT levels, a key pro-survival signal, were found to be very low in most high-grade lymphoma cell lines and primary NHL specimens, in contrast to the high levels reported in carcinomas. Low AKT activity may bias lymphoma cells toward apoptosis in response to SGN-40 signaling. To augment SGN-40-induced cell killing, in vitro combination studies with chemotherapeutics have been performed using the Ramos, RL, and HT NHL lines. We now report that SGN-40 has additive activity when combined with cisplatin, melphalan, or mitoxantrone and is synergistic with bleomycin. Furthermore, in vivo activity of combination therapy has been demonstrated in a subcutaneous Ramos lymphoma xenograft model. While tumor growth was delayed by either CHOP (cyclophosphamide, adriamycin, vincristine, prednisone) or SGN-40 alone, the combination of SGN-40 and CHOP was significantly more active. Our results suggest that SGN-40 can be combined with standard lymphoma therapies resulting in improved therapeutic efficacy, and provide a rationale for combination clinical trials involving SGN-40. The molecular mechanisms through which standard chemotherapeutic agents enhance SGN-40-mediated cell killing in target lymphoma B cells are currently being investigated.

The BCL6 gene in B-cell lymphomas with 3q27 translocations is expressed mainly from the rearranged allele irrespective of the partner gene.

The BCL6 gene, which functions as a transcription repressor, is the target of multiple chromosomal translocations in non-Hodgkin's lymphomas (NHL). These translocations occur in the nontranslated region of the BCL6 gene, juxtaposing regulatory sequences of the diverse partner genes to the open reading frame of the BCL6 gene and thus are thought to deregulate BCL6 gene expression. The levels of expression of the BCL6 gene and protein have been demonstrated to predict the clinical outcome of diffuse large B-cell lymphomas. By contrast, the prognostic significance of BCL6 gene translocations is unclear. In this study we have sought an explanation for this apparent discrepancy. We examined tumors with a variety of different BCL6 translocations and therefore with a variety of potentially substituted promoters. We found no increase in total BCL6 mRNA levels in the NHL specimens harboring BCL6 gene translocation. Indeed, some of these tumors expressed relatively low quantities of the BCL6 mRNA. We also sought to determine whether BCL6 transcription occurs from the rearranged or from the normal untranslocated allele in these tumors. We demonstrate that lymphoma cell lines and majority of NHL tumor specimens expressed BCL6 mRNA predominantly from the rearranged allele that may come under the control of various partner gene promoters. However, few NHL tumors with BCL6 gene translocations expressed BCL6 mRNA equally from the rearranged and the nonrearranged alleles. Neither the nature of the substituted promoters nor the presence of activating mutations in the BCL6 regulatory sequences correlated with the allelic expression of the BCL6 gene in these tumors.

Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies.

Real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) is a powerful method for measurement of gene expression for diagnostic and prognostic studies of non-Hodgkin's lymphomas (NHL). In order for this technique to gain wide applicability, it is critically important to establish a uniform method for normalization of RNA input. In this study, we have determined the best method to quantify the RNA/cDNA input per reaction and searched for the most useful endogenous control genes for normalization of the measurements, based on their abundance and lowest variability between different types of lymphoid cells. To accomplish these aims, we have analyzed the RNA expression of 11 potential endogenous control genes (glyceraldehyde-3-phosphate dehydrogenase, beta-actin, peptidylprolyl isomerase A, beta 2 microglobulin, protein kinase cGMP-dependent, type I, hypoxanthine phosphoribosyltransferase 1, TATA box binding protein, transferrin receptor, large ribosomal protein, beta-glucoronidase and 18S ribosomal RNA). In all, 12 different B- and T-cell lymphoma/leukemia cell lines, 80 B- and T-cell NHL specimens, and resting and activated normal B and T lymphocytes were screened. Normalization of the nucleic acid input by spectrophotometric OD(260) measurement of RNA proved more reliable than spectrophotometric or fluorometric measurements of cDNA or than electrophoretic estimation of the ribosomal and mRNA fractions. The protein kinase cGMP-dependent, type I (PRKG1) and the TBP genes were expressed at common abundance and exhibited the lowest variability among the cell specimens. We suggest that for further lymphoma studies based on the real-time RT-PCR quantification of gene expression, that RNA input in each reaction be equalized between the specimens by spectrophotometric OD(260) measurements. The expression of the gene of interest in different samples should be normalized by concomitant measurement of the PRKG1 and/or the TBP gene products.

Expression of the monomeric 67- kd lamininbinding protein in human lymphomas as defined by MLuC5 monoclonal antibody and paraffin section immunohistochemistry

Interactions between cancer cells and laminin, a major component of basement membranes, are mediated through a large variety of cell surface proteins designated as laminin receptors. Among the above proteins, a 67-kd monomeric high affinity laminin receptor (67 LR) has long been suspected to be involved in tumor progression. In this study we wished to establish whether the 67 LR molecule is detectable on tumor cells of Hodgkin's disease (HD) and non-Hodgkin's lymphomas (NHLs), to define its pattern of expression, and to assess the potential utility of 67 LR in differentiating these pathological entities. Morphological and immunohistological studies were performed on 85 specimens of HD and a series of 334 NHL specimens, including anaplastic large cell (ALC) (CD30-positive) lymphomas (73 specimens). For immunohistochemical assessment of the 67 LR we used the monoclonal antibody (MoAb) MLuC5 directed against the 67-kd laminin receptor on paraffin-embedded sections. Reed-Sternberg cells reacted with MLuC5 MoAb in four of 85 (4.7%) HD specimens. Among the NHL specimens, a MLuC5-positive reaction was expressed in 3.3% of B-cell lymphomas. They all belonged to the high grade subtypes. On the other hand, a MLuC5-positive reaction was detected in none of the T-cell lymphomas tested. In contrast to the results obtained with the other NHLs, in 30.2% of ALC (CD30-positive) lymphoma specimens, tumor cells reacted with MLuC5 MoAb. MLuC5-expressing ALC (CD30-positive) lymphoma cells were of either T-cell (six of 17 specimens), B-cell (three of 25 specimens), or undetermined phenotype (10 of 31 specimens). Our investigation has shown that 67 LR as shown by MLuC5 MoAb is detectable only in neoplastic cells of a fraction of ALC (CD30-positive) lymphomas and small subsets of B-cell high grade NHLs and HD. The restricted expression of the 67 LR molecule to ALC (CD30-positive) lymphomas provides a potential tool for the phenotypic separation of this pathological entity from HD and other lymphomas. Whether the detection of the 67 LR expression in these lymphoma subsets may be related to the aggressiveness of the disease remains to be ascertained.

Geographic sequence variation of latent membrane protein 1 gene of Epstein-Barr virus in Hodgkin's lymphomas.

To assess the role of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene in the development of Hodgkin's lymphoma (HL), the polymorphism of this gene in EBV isolates from different geographic locations was analyzed. A 497 bp fragment spanning LMP1 gene exons 1 and 2 was amplified by polymerase chain reaction (PCR), using a primer pair bracketing a Xhol restriction site. PCR products were subjected to Xhol digestion and to DNA sequencing analysis. Twenty-five HL biopsy specimens from the United States and five HL and four non-Hodgkin's lymphoma (NHL) biopsy specimens from Italy were examined. Eighty percent of LMP1-positive samples (12 of 15) from the United States maintained the Xhol restriction site and the remaining 20% partially lost the Xhol site. One of four EBV-positive HL and one of the three EBV-positive NHL specimens from Italy lost the restriction site. The other three EBV-positive HL DNAs were partially cut by Xhol. Direct DNA sequencing analysis revealed that those Italian samples not digested by Xhol were due to a G to C transversion at the first base of codon 18, resulting in the change of glycine to arginine. Those DNA samples partially cut by Xhol were due to a mixture of G/C at the same location. In contrast, those partially digested American HL DNAs had a mixture of G/T at the second base of codon 17.(ABSTRACT TRUNCATED AT 250 WORDS)

Engraftment of human non-hodgkin lymphomas in mice with severe combined immunodeficiency

Malignant non-Hodgkin lymphoma (NHL) is one of the most difficult neoplasms to transplant into nude mice. Mice with severe combined immunodeficiency (SCID) accept various human cancers much more efficiently than do nude mice. The authors investigated whether SCID mice could be used as convenient hosts in which to grow human NHL in vivo. Fifty NHL specimens were engrafted into SCID mice. The original specimens and the tumors that developed in SCID mice were studied immunohistologically and by Southern blot analysis to clarify their clonal identity and to determine if they were Epstein-Barr virus (EBV)-transformed B cell proliferations. SCID tumors developed from 23 of 50 NHL specimens. Ten tumors were identical immunophenotypically and, partly, genotypically to the original NHL, showing that the original NHL grew in the SCID mice. B-cell NHL rather than T-cell NHL and high-grade rather than low-grade malignancy groups were much more easily heterotransplanted. Most of the heterotransplanted NHL were maintained by successive transplantation. In two other SCID tumors, the original NHL clones and a newly developed B-cell clone coexisted. The remaining 11 SCID tumors were composed of newly developed clones. The latter 13 tumors were shown to be human cells of B-cell lineage bearing EBV latent proteins--latent membrane protein 1 and EB nuclear antigen 2--suggesting that they originated from EBV-infected B-cells that were present in the original tumor tissues. SCID mice accept human NHL far more efficiently than do nude mice. However, frequent occurrence of spontaneous EBV-associated B-cell proliferation must be kept in mind.