Frederike von Bonin

Effect of Interleukin-10 Gene Polymorphisms on Clinical Outcome of Patients with Aggressive Non-Hodgkin's Lymphoma: An Exploratory Study

Purpose: Current chemotherapy can achieve high response rates in aggressive non-Hodgkins lymphoma (NHL), but the factors that influence regression and survival remain unknown. The present exploratory study tested the hypothesis whether interleukin-10 (IL-10) polymorphisms predict clinical outcome, leukocytopenia, or infectivity during therapy. IL-10 was chosen because immune alterations are a major risk factor for NHL, and IL-10 is a cytokine involved in inflammatory processes associated with clinical outcome. Experimental Design: Five hundred patients with aggressive NHL treated with CHOP/CHOEP were analyzed for IL-10 gene polymorphisms, including distal loci -7400InDel, -6752AT (rs6676671), and -6208CG (rs10494879) in comparison with proximal loci -3538AT (rs1800890), -1087AG (rs1800896), and -597AC (rs1800872) according to the incidence and outcome of the lymphoma. Results: No differences in allele frequencies or haplotypes were found comparing a cohort of patients with aggressive NHL/diffuse large B-cell lymphoma with a healthy control group. Patients with aggressive NHL characterized by IL-10-7400DelDel had shorter overall survival periods compared with the other genotypes (P = 0.004). The 3-year rate is 43.4% for IL-10-7400DelDel and 73.4% for IL-10-7400InIn and IL-10-7400InDel together. A significant increased risk for event-free survival is found for carriers of the genotype IL-10-6752TT-6208CC-3538AA (P = 0.047). Multivariate analysis of IL-10-7400 gene variation in relation to overall survival adjusted to international prognostic index revealed a relative risk of 1.9 for carriers of IL-10-7400DelDel (P = 0.037). No associations were found analyzing diffuse large B-cell lymphoma patients separately. Conclusion: Our results indicate that IL-10 gene variations could be associated to the clinical course of aggressive NHL, which points out the importance of host factors and respective genetic elements for treatment response.

The MICA-129 dimorphism affects NKG2D signaling and outcome of hematopoietic stem cell transplantation

The MHC class I chain‐related molecule A (MICA) is a highly polymorphic ligand for the activating natural killer (NK)‐cell receptor NKG2D. A single nucleotide polymorphism causes a valine to methionine exchange at position 129. Presence of a MICA‐129Met allele in patients (n = 452) undergoing hematopoietic stem cell transplantation (HSCT) increased the chance of overall survival (hazard ratio [HR] = 0.77, P = 0.0445) and reduced the risk to die due to acute graft‐versus‐host disease (aGVHD) (odds ratio [OR] = 0.57, P = 0.0400) although homozygous carriers had an increased risk to experience this complication (OR = 1.92, P = 0.0371). Overall survival of MICA‐129Val/Val genotype carriers was improved when treated with anti‐thymocyte globulin (HR = 0.54, P = 0.0166). Functionally, the MICA‐129Met isoform was characterized by stronger NKG2D signaling, triggering more NK‐cell cytotoxicity and interferon‐γ release, and faster co‐stimulation of CD8+ T cells. The MICA‐129Met variant also induced a faster and stronger down‐regulation of NKG2D on NK and CD8+ T cells than the MICA‐129Val isoform. The reduced cell surface expression of NKG2D in response to engagement by MICA‐129Met variants appeared to reduce the severity of aGVHD.

HSP90 is essential for Jak-STAT signaling in classical Hodgkin Lymphoma cells

In classical Hodgkin lymphoma (cHL) chemotherapeutic regimens are associated with stagnant rates of secondary malignancies requiring the development of new therapeutic strategies. We and others have shown that permanently activated Signal Transducer and Activator of Transcription (STAT) molecules are essential for cHL cells. Recently an overexpression of heat-shock protein 90 (HSP90) in cHL cells has been shown and inhibition of HSP90 seems to affect cHL cell survival. Here we analysed the effects of HSP90 inhibition by geldanamycin derivative 17-AAG or RNA interference (RNAi) on aberrant Jak-STAT signaling in cHL cells. Treatment of cHL cell lines with 17-AAG led to reduced cell proliferation and a complete inhibition of STAT1, -3, -5 and -6 tyrosine phosphorylation probably as a result of reduced protein expression of Janus kinases (Jaks). RNAi-mediated inhibition of HSP90 showed similar effects on Jak-STAT signaling in L428 cHL cells. These results suggest a central role of HSP90 in permanently activated Jak-STAT signaling in cHL cells. Therapeutics targeting HSP90 may be a promising strategy in cHL and other cancer entities associated with deregulated Jak-STAT pathway activation.

Dysfunctional p53 deletion mutants in cell lines derived from Hodgkin's lymphoma

Classical Hodgkins lymphoma (cHL) is a distinct malignancy of the immune system. Despite the progress made in the understanding of the pathology of cHL, the transforming events remain to be elucidated. It has been proposed that mutations in the TP53 gene in biopsy material as well as cell lines derived from cHL are rare and therefore not notably involved in the pathogenesis of the malignant H&RS cells. Re-evaluating the expression in cHL-derived cell lines, we found that in 3/6 of these cell lines, TP53 transcripts are characterized by deletions within exon 4 (L428 cells) and nearly a complete loss of exons 10 – 11 (L1236) or exons 8 – 11 (HDLM-2), respectively. These changes were found in otherwise rarely mutated regions of TP53. Cell lines L1236 and HDLM-2 harbour fusions with alu-repeats in their TP53 mRNA 3′-ends, resulting in the carboxyterminal truncation and loss of the transcriptional activity of p53. Transcriptional inactivity was also found for p53 in L428 cells. This study characterizes mutations in TP53 transcripts within cHL cell lines with associated functional defects in the resulting p53 proteins and therefore reintroduces the concept that mutations of TP53 might be involved in the pathogenesis of Hodgkins lymphoma.

Diffuse large B-cell lymphoma patient-derived xenograft models capture the molecular and biological heterogeneity of the disease

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease defined by transcriptional classifications, specific signaling and survival pathways, and multiple low-frequency genetic alterations. Preclinical model systems that capture the genetic and functional heterogeneity of DLBCL are urgently needed. Here, we generated and characterized a panel of large B-cell lymphoma (LBCL) patient-derived xenograft (PDX) models, including 8 that reflect the immunophenotypic, transcriptional, genetic, and functional heterogeneity of primary DLBCL and 1 that is a plasmablastic lymphoma. All LBCL PDX models were subjected to whole-transcriptome sequencing to classify cell of origin and consensus clustering classification (CCC) subtypes. Mutations and chromosomal rearrangements were evaluated by whole-exome sequencing with an extended bait set. Six of the 8 DLBCL models were activated B-cell (ABC)-type tumors that exhibited ABC-associated mutations such as MYD88, CD79B, CARD11, and PIM1. The remaining 2 DLBCL models were germinal B-cell type, with characteristic alterations of GNA13, CREBBP, and EZH2, and chromosomal translocations involving IgH and either BCL2 or MYC Only 25% of the DLBCL PDX models harbored inactivating TP53 mutations, whereas 75% exhibited copy number alterations of TP53 or its upstream modifier, CDKN2A, consistent with the reported incidence and type of p53 pathway alterations in primary DLBCL. By CCC criteria, 6 of 8 DLBCL PDX models were B-cell receptor (BCR)-type tumors that exhibited selective surface immunoglobulin expression and sensitivity to entospletinib, a recently developed spleen tyrosine kinase inhibitor. In summary, we have established and characterized faithful PDX models of DLBCL and demonstrated their usefulness in functional analyses of proximal BCR pathway inhibition.

Aberrant lymphocyte enhancer-binding factor 1 expression is characteristic for sporadic Burkitt's lymphoma

Burkitts lymphoma (BL) is a highly malignant, aggressive non-Hodgkins lymphoma derived from germinal center B cells. Recently, global gene expression profiling of patient samples led to a molecular definition of BL with lymphocyte enhancer-binding factor 1 (LEF1) as a signature gene. Herein, we report the expression of nucleic LEF1 in 15 of 18 patients with BL and the identification of LEF1 target genes. Germinal center B cells were devoid of detectable nuclear LEF1 expression, as were mantle cell lymphoma (0 of 5), marginal zone lymphoma (0 of 6), follicular lymphoma (0 of 12), and diffuse large B-cell lymphoma (1 of 31). Whole-genome gene expression profiling after transient knockdown of LEF1 in BL cell lines identified new LEF1 target genes; these LEF1 targets are enriched with genes associated with cancers. The expression of LEF1 and LEF1-regulated genes in primary BL suggests that LEF1 is not only aberrantly expressed but also transcriptionally active. This study supports a functionally important role for LEF1 and its target genes in BLs.

Global gene expression changes of in vitro stimulated human transformed germinal centre B cells as surrogate for oncogenic pathway activation in individual aggressive B cell lymphomas

BackgroundAggressive Non-Hodgkin lymphomas (NHL) are a group of lymphomas derived from germinal centre B cells which display a heterogeneous pattern of oncogenic pathway activation. We postulate that specific immune response associated signalling, affecting gene transcription networks, may be associated with the activation of different oncogenic pathways in aggressive Non-Hodgkin lymphomas (NHL).MethodologyThe B cell receptor (BCR), CD40, B-cell activating factor (BAFF)-receptors and Interleukin (IL) 21 receptor and Toll like receptor 4 (TLR4) were stimulated in human transformed germinal centre B cells by treatment with anti IgM F(ab)2-fragments, CD40L, BAFF, IL21 and LPS respectively. The changes in gene expression following the activation of Jak/STAT, NF-кB, MAPK, Ca2+ and PI3K signalling triggered by these stimuli was assessed using microarray analysis. The expression of top 100 genes which had a change in gene expression following stimulation was investigated in gene expression profiles of patients with Aggressive non-Hodgkin Lymphoma (NHL).Resultsα IgM stimulation led to the largest number of changes in gene expression, affecting overall 6596 genes. While CD40L stimulation changed the expression of 1194 genes and IL21 stimulation affected 902 genes, only 283 and 129 genes were modulated by lipopolysaccharide or BAFF receptor stimulation, respectively. Interestingly, genes associated with a Burkitt-like phenotype, such as MYC, BCL6 or LEF1, were affected by αIgM. Unique and shared gene expression was delineated. NHL-patients were sorted according to their similarity in the expression of TOP100 affected genes to stimulated transformed germinal centre B cells The αIgM gene module discriminated individual DLBCL in a similar manner to CD40L or IL21 gene modules. DLBCLs with low module activation often carry chromosomal MYC aberrations. DLBCLs with high module activation show strong expression of genes involved in cell-cell communication, immune responses or negative feedback loops. Using chemical inhibitors for selected kinases we show that mitogen activated protein kinase- and phosphoinositide 3 kinase-signalling are dominantly involved in regulating genes included in the αIgM gene module.ConclusionWe provide an in vitro model system to investigate pathway activation in lymphomas. We defined the extent to which different immune response associated pathways are responsible for differences in gene expression which distinguish individual DLBCL cases. Our results support the view that tonic or constitutively active MAPK/ERK pathways are an important part of oncogenic signalling in NHL. The experimental model can now be applied to study the therapeutic potential of deregulated oncogenic pathways and to develop individual treatment strategies for lymphoma patients.

Circulating levels of TNF- receptor II are prognostic for patients with peripheral T-cell Non-Hodgkin lymphoma

Purpose: Peripheral T-cell non–Hodgkin lymphomas (T-NHL) represent a small but heterogeneous and clinically aggressive subset of NHLs with a poor outcome. Cytokines or their receptors might be associated with the clinical outcome of these lymphomas. Therefore, we tested whether gene variations and serum levels of soluble TNF receptor (TNFR)I (sTNFRI), sTNFRII, interleukin (IL)-10, or sIL-4R are predictive for treatment response in T-NHLs. Experimental Design: Peripheral blood DNA from 117 patients with T-NHL treated in prospective clinical trials was subjected to genotyping analysis. Whenever possible, pretreatment sera were obtained, and circulating levels of sTNFRI, sTNFRII, IL-10, and sIL-4R were determined with a specific capture enzyme-linked immunoassay. Results: Patients characterized by TNFRI-609GG (rs4149570) showed a trend toward better event free survival [EFS; univariate: P = 0.041; multivariate: HR, 1.76; confidence interval (CI), 0.99–3.14 with P = 0.056]. A protective role of IL-10–1087A, −824T, and −597A reported in another study was not confirmed in our cohort. Patients with circulating levels of soluble TNFRII ≥2.16 ng/mL had a 2.07-fold increased relative risk for shorter overall survival (OS; univariate: P = 0.0034; multivariate: HR, 2.07; CI, 0.92–4.70 with P = 0.081) and a 2.49-fold higher risk for shorter EFS (univariate: P = 0.00068; multivariate: HR, 2.49; CI, 1.22–5.08 with P = 0.012). Elevations of circulating levels of sTNFRI, IL-10, and sIL-4R are frequent, but the clinical response in these patients is not significantly different. Conclusions: Our findings suggest a critical role for TNF-TNFR signaling for the clinical outcome of patients with peripheral T-NHLs. Clin Cancer Res; 18(13); 3637–47. ©2012 AACR.

Immunophenotypic and genetic characterization of a CD8 positive Mantle Cell Lymphoma in a patient with concomitant Mycosis Fungoides

Abstract:  Mantle cell lymphoma (MCL) is immunophenotypically characterized by cell surface co‐expression of CD19, CD20, CD5, IgM and FMC7. However, the concomitant presence of other antigens distinctive of a particular leukocyte subset, e.g. T‐lymphocytes, is an exceptional finding in MCL. Here, the first case of a blastic MCL in leukaemic phase with aberrant expression of the T‐cell associated antigen CD8 occurring in a patient with concomitant Mycosis fungoides is described. Comprehensive immunophenotypic analysis showed that the MCL cells expressed the typical B‐lymphocytic markers, were CD5 and CD8 positive, but did not express other T‐cell proteins, such as CD2, CD3, CD4, CD7, TCRαβ and TCRγδ. The MCL cells expressed both CD8α and CD8β chains indicating cell surface presence of CD8αβ heterodimers. Intriguingly, expression of the cytotoxic enzymes perforin and granzyme A was detected by RT‐PCR. Cytogenetic and molecular genetic analysis of the lymphoma cells confirmed cyclin D1 overexpression secondary to the t(11;14)(q13;32) chromosomal translocation. Furthermore, trisomy 11, trisomy 14 and extra copies of t(11;14) translocated chromosomes were detected in sub clones of the analyzed MCL cells. Clinically, an aggressive course of disease including cerebral lymphoma involvement was noted in the reported patient. Hence, systematic studies addressing the incidence, biology and clinical behavior of this form of MCL seem to be justified in future.

Specific tumor-stroma interactions of EBV-positive Burkitt's lymphoma cells in the chick chorioallantoic membrane

BackgroundBurkitts lymphoma (BL) is an aggressive Non-Hodgkin lymphoma. Epstein-Barr Virus (EBV) is able to transform B cells and is a causative infectious agent in BL. The precise role of EBV in lymphoma progression is still unclear. Most investigations have concentrated on cell autonomous functions of EBV in B cells. Functions of the local environment in BL progression have rarely been studied, mainly due to the lack of appropriate in vivo models. Therefore, we inoculated different human BL cell-lines onto the chorioallantoic membrane (CAM) of embryonic day 10 (ED10) chick embryos and re-incubated until ED14 and ED17.ResultsAll cell-lines formed solid tumors. However, we observed strong differences in the behavior of EBV+ and EBV- cell-lines. Tumor borders of EBV+ cells were very fuzzy and numerous cells migrated into the CAM. In EBV- tumors, the borders were much better defined. In contrast to EBV- cells, the EBV+ cells induced massive immigration of chick leukocytes at the tumor borders and the development of granulation tissue with large numbers of blood vessels and lymphatics, although the expression of pro- and anti-angiogenic forms of Vascular Endothelial Growth Factors/receptors was the same in all BL cell-lines tested. The EBV+ cell-lines massively disseminated via the lymphatics and completely occluded them.ConclusionsOur data suggest that the EBV+ cells attract pro-angiogenic leukocytes, which then induce secondary tumor-stroma interactions contributing to the progression of BL. We show that the CAM is a highly suitable in vivo model to study the differential behavior of BL cell-lines.