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Classical Hodgkin lymphoma (cHL) has a unique pathological feature characterized by a minority of malignant Hodgkin Reed-Sternberg (H-RS) cells surrounded by numerous inflammatory cells. Cysteinyl-leukotrienes (CysLTs) are produced by eosinophils, macrophages and mast cells in the HL tumor microenvironment. In the present study we have explored the signal transduction pathways leading to leukotriene (LT) D4 induced expression of cytokines in the Hodgkin lymphoma cell line L1236 and KM-H2. Stimulation of L1236 and KM-H2 cells with LTD4 led to a concentration- and time-dependent increase at the transcriptional level of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3) and CCL4. The expression of several transcription factors was induced upon stimulation of Hodgkin cell lines with LTD4. Among these, EGR-1 was required for cytokine production. Inhibition of EGR-1 expression using shEGR-1 transduced by lentivirus led to suppression of the expression of TNF-α and IL-6. The effect of LTD4 on the expression of transcription factors and cytokines were also blocked by the specific CysLT1 receptor antagonist zafirlukast. These results demonstrate that EGR-1 plays a critical role in LTD4-induced cytokine transcription in Hodgkin cell lines.

Patients with Hodgkin lymphoma (HL) have a well-characterized immune deficiency of T cell function, originally identified by increased susceptibility to certain infections. Epidemiological evidence has long pointed to infectious etiologies in younger HL patients. With the aim of expanding our knowledge on the potential role of pre-existing immune deficiency in HL and an infectious/inflammatory etiology, we conducted a comprehensive population-based case–control study in HL patients diagnosed in Sweden in the period 1965–2004, and their matched controls. In a large population-based study including 7,414 HL patients and 29,240 matched controls, we evaluated the subsequent risk of HL in relation to a broad range of infectious and inflammatory conditions, using unconditional logistic regression. A previous history of any reported infection was associated with an 11 % increased risk of HL (P < 0.05). More specifically, we found sinusitis (odds ratio = 1.81; 95 % confidence interval = 1.06–3.07), tuberculosis (1.76; 1.01–3.07), encephalitis (7.88; 1.97–31.5), and herpes zoster (2.20; 1.11–4.35) to be associated with excess HL risk. A personal prior history of chronic inflammatory condition was not associated with an increased risk of HL (0.94; 0.71–1.14). Our results suggest that underlying immune deficiency is a primary phenomenon in HL. Alternatively, certain infectious agents may be potential HL triggers.

BACKGROUND Studies of mechanisms mediating resistance to chemotherapy led to the discovery of the multidrug transporter ABCB1 (ATP-binding cassette, subfamily B, member 1), often expressed in leukemic cells of patients with acute myeloid leukemia (AML). Most clinical trials evaluating the strategy of inhibiting efflux-mediated chemotherapeutic resistance have been unsuccessful, clearly indicating the need for a better approach. METHODS This study investigated the clinical relevance of 380 genes whose expression has been shown to affect the response to chemotherapy, mostly through in vitro studies, in 11 paired samples obtained at AML diagnosis and at relapse. The expression profiling of these 380 genes was performed using TaqMan-based quantitative reverse-transcription polymerase chain reaction. Patients had a median age of 58 years at diagnosis, a median duration of complete remission of 284.5 days, and a median overall survival of 563 days. Cytogenetic abnormalities were detected at diagnosis in 4 patients, whereas 5 displayed a normal karyotype and 2 were not investigated. RESULTS Hierarchical clustering shows that samples taken at diagnosis and relapse clustered in pairs for 6 patients of the 11 studied, suggesting recurrence of the same leukemic blast, whereas for the other 5 patients, the data indicate their relapse blasts arose from different origins. A patient-by-patient analysis of the paired samples led to the striking observation that each had a unique gene signature representing different mechanisms of resistance. CONCLUSIONS The data underline the need for personalized molecular analysis to tailor treatment for patients with AML. Cancer 2013;119:3076—3083. © 2013 American Cancer Society.

Purpose Chronic myeloid leukemia (CML) management changed dramatically with the development of imatinib mesylate (IM), the first tyrosine kinase inhibitor targeting the BCR-ABL1 oncoprotein. In Sweden, the drug was approved in November 2001. We report relative survival (RS) of patients with CML diagnosed during a 36-year period. Patients and Methods Using data from the population-based Swedish Cancer Registry and population life tables, we estimated RS for all patients diagnosed with CML from 1973 to 2008 (n = 3,173; 1,796 males and 1,377 females; median age, 62 years). Patients were categorized into five age groups and five calendar periods, the last being 2001 to 2008. Information on use of upfront IM was collected from the Swedish CML registry. Results Relative survival improved with each calendar period, with the greatest improvement between 1994-2000 and 2001-2008. Five-year cumulative relative survival ratios (95% Cls) were 0.21 (0.17 to 0.24) for patients diagnosed 1973-1979, 0.54 (0.50 to 0.58) for 1994-2000, and 0.80 (0.75 to 0.83) for 2001-2008. This improvement was confined to patients younger than 79 years of age. Five-year RSRs for patients diagnosed from 2001 to 2008 were 0.91 (95% CI, 0.85 to 0.94) and 0.25 (95% CI, 0.10 to 0.47) for patients younger than 50 and older than 79 years, respectively. Men had inferior outcome. Upfront overall use of IM increased from 40% (2002) to 84% (2006). Only 18% of patients older than 80 years of age received IM as first-line therapy. Conclusion This large population-based study shows a major improvement in outcome of patients with CML up to 79 years of age diagnosed from 2001 to 2008, mainly caused by an increasing use of IM. The elderly still have poorer outcome, partly because of a limited use of IM.

Young-adult-onset (15–44 years of age) Hodgkin lymphoma (HL) is believed to arise as a consequence of late primary infection in susceptible individuals. The properties of this susceptibility remain little understood. We have previously reported an increased occurrence of HL in patients with rheumatoid arthritis and among their offspring, suggesting that susceptibility to autoimmunity might be of importance also in the pathogenesis of HL. To explore this hypothesis, we assessed the association of personal and family history of diabetes mellitus, with risk of subsequent HL in a population-based case-control study, including as cases all individuals diagnosed with HL above 15 years of age 1964–1999 (n=6,873) in Sweden, and matched population controls (n=12,565). First-degree relatives of cases and controls were identified through linkage with the Multi-generation Register. We identified discharges listing diabetes mellitus through linkage with the Inpatient Register (1964–2000). We used odds ratios (OR) as measures of relative risk. Cases with young-adult-onset HL were less likely to have a personal (OR=0.5, 95% CI 0.2–1.1) or family (OR=0.7, 95% CI 0.6–0.8) history of diabetes mellitus. In contrast, HL diagnosed at older ages was neither associated with a personal (OR=1.0) nor family (OR=1.0) history of diabetes mellitus. These findings suggests that characteristics of the immune system associated with conditions such as diabetes mellitus type I are of importance in the pathogenesis of young-adult-onset HL. © 2005 Wiley-Liss, Inc.

Summary. We investigated whether the described immune evasion of Epstein–Barr virus (EBV)-infected malignant Hodgkin and Reed-Sternberg (HRS) cells in Hodgkin's disease (HD) is paralleled by a disturbed expression of the signal transduction molecule ζ associated with CD3 and CD16 in tumour-associated T lymphocytes (TAL). Flow cytometric analysis revealed a significantly lower ζ expression in CD3+/4+, CD3+/8+ and CD16+ patient peripheral blood lymphocytes (PBL; n = 10) compared with normal donor PBLs (n = 11). When patient PBLs were compared with the corresponding TAL, the latter showed a significantly higher (CD3+/4+) or equal (CD3+/8+) ζ expression. The EBV status of the tumours did not correlate with ζ expression in the TAL. Immunohistochemical staining revealed ζ-positive lymphocytes among the adjacent bystander cells of the HRS cells in all analysed tumours (n = 8), irrespective of tumour EBV status. In conclusion, these results do not support downregulation of ζ in TAL as a critical mechanism contributing specifically to the immune escape of EBV+ HRS cells.

Regulation of cytokine levels has been shown to be under genetic control through the coding and promoter sequences of genetic polymorphisms. We elucidated the prevalence of a previously described G to A transition polymorphism at position −308 of the tumour necrosis factor-α (TNF-α) promoter region in a population of patients with Hodgkin's disease (HD) (n = 36) and chronic lymphocytic leukaemia (CLL) (n = 49) and healthy volunteers (n = 51). The DNA fragment containing this polymorphism was amplified by PCR and sequenced by solid-phase minisequencing. The frequency of the TNF-α promoter polymorphism was not significantly different between CLL patients and HD patients compared to controls.

Larvae of the Northern pine processionary moth (Thaumetopoea pinivora, TP) carry microscopic needles (setae), which by penetrating skin and mucous membranes, may cause inflammatory/immune derived symptoms in man. In the present study the stimulatory effects of setae on human blood lymphocytes in vitro was investigated. Blood mononuclear cells were separated from venous blood or buffy coat of ten healthy individuals, six previously exposed to setae and four with no known exposure. Lymphoproliferation was measured as uptake of 3H-thymidine. Setae were prepared from TP larvae. Setae and saline setae extracts stimulated proliferation of T-lymphocytes in the presence of monocytic cells. Stimulation was pronounced in cells from persons who had been exposed to setae, and weak in cells from non-exposed donors. Chitin also induced lymphocyte proliferation in most donors, but to a lesser extent and independently of donor's previous exposure to setae. In conclusion, setae contain molecules that in the presence of monocytes activate human T-lymphocytes to proliferation. The antigenic nature of stimulatory molecules was supported by the significantly stronger lymphocyte response in persons previously exposed to setae than in non-exposed donors. The nature of such molecules remains to be defined.

On May 10, 2012, the European Commission issued a conditional marketing authorization valid throughout the European Union for pixantrone for the treatment of adult patients with multiply relapsed or refractory aggressive non-Hodgkin's B-cell lymphoma (NHL). Pixantrone is a cytotoxic aza-anthracenedione that directly alkylates DNA-forming stable DNA adducts and cross-strand breaks. The recommended dose of pixantrone is 50 mg/m(2) administered on days 1, 8, and 15 of each 28-day cycle for up to 6 cycles. In the main study submitted for this application, a significant difference in response rate (proportion of complete responses and unconfirmed complete responses) was observed in favor of pixantrone (20.0% vs. 5.7% for pixantrone and physician's best choice, respectively), supported by the results of secondary endpoints of median progression-free and overall survival times (increase of 2.7 and 2.6 months, respectively). The most common side effects with pixantrone were bone marrow suppression (particularly of the neutrophil lineage) nausea, vomiting, and asthenia. This article summarizes the scientific review of the application leading to approval in the European Union. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the European Medicines Agency website (http://www.ema.europa.eu).

Interleukin-10 (IL-10) has been shown in vitro to inhibit survival and spontaneous DNA synthesis in B-cell chronic lymphocytic leukaemia (B-CELL) cells by induction of programmed cell death. We have analysed the presence of mRNA transcripts for IL-10 in purified B-CLL cells from 35 patients by RT-PCR. Transcripts for IL-10 were detected in 11/20 patients with non-progressive disease. In cell preparations from patients with progressive B-CLL IL-10 mRNA were detected in only 2/15 samples (P < or = 0.01). The Epstein-Barr virus status of the cells did not account for the difference in IL-10 mRNA expression observed between the two groups of patients. Thus, IL-10 mRNA expression in leukaemic cells from patients with B-CLL was strongly associated with non-progressive disease. This finding may support other observations suggesting that IL-10 might be a candidate for immune therapy of progressive B-CLL.

In recent decades, attention has focused on reducing long-term, treatment-related morbidity and mortality in Hodgkin lymphoma (HL). In the present study, we looked for trends in relative survival for all patients diagnosed with HL in Sweden from 1973-2009 (N = 6949; 3985 men and 2964 women; median age, 45 years) and followed up for death until the end of 2010. Patients were categorized into 6 age groups and 5 calendar periods (1973-1979, 1980-1986, 1987-1994, 1994-2000, and 2001-2009). Relative survival improved in all age groups, with the greatest improvement in patients 51-65 years of age (P < .0005). A plateau in relative survival was observed in patients below 65 years of age during the last calendar period, suggesting a reduced long-term, treatment-related mortality. The 10-year relative survival for patients diagnosed in 2000-2009 was 0.95, 0.96, 0.93, 0.80, and 0.44 for the age groups 0-18, 19-35, 36-50, 51-65, and 66-80, respectively. Therefore, despite progress, age at diagnosis remains an important prognostic factor (P < .0005). Advances in therapy for patients with limited and advanced-stage HL have contributed to an increasing cure rate. In addition, our findings support that long-term mortality of HL therapy has decreased. Elderly HL patients still do poorly, and targeted treatment options associated with fewer side effects will advance the clinical HL field.

Classical Hodgkin lymphoma (cHL) is characterized histologically by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by abundant inflammatory cells, generally believed to be of major importance in the pathophysiology of the disease. Here, we present data that link inflammatory cell-derived arachidonic acid metabolites, the cysteinyl leukotrienes (CysLT), to the pathogenesis of cHL. Two HL cell lines, L1236 and KMH2, were shown to express functional CysLT1 receptors, responding with a robust calcium signal upon leukotriene (LT) D4 challenge. LTD4 stimulated protein release of tumor necrosis factor-α, interleukin-6 and -8 by L1236 cells and interleukin-8 by KMH2 cells. Importantly, all these LTD4-induced effects were blocked by the CysLT1 receptor-specific antagonist zafirlukast. Immunohistochemical studies of cHL biopsies and microarray analysis of microdissected cells revealed that the CysLT1 receptor is expressed also by primary Hodgkin Reed-Sternberg cells. As these cells are surrounded by CysLT-producing eosinophils, macrophages and mast cells, our results suggest the CysLTs as mediators in the pathogenesis of cHL, contributing to the aberrant cytokine network of this lymphoma. © 2008 Wiley-Liss, Inc.

Classical Hodgkin lymphoma has unique clinical and pathological features and tumour tissue is characterized by a minority of malignant Hodgkin Reed-Sternberg cells surrounded by inflammatory cells. In the present study, we report that the Hodgkin lymphoma-derived cell line L1236 has high expression of 15-lipoxygenase-1 and that these cells readily convert arachidonic acid to eoxin C(4), eoxin D(4) and eoxin E(4). These mediators were only recently discovered in human eosinophils and mast cells and found to be potent proinflammatory mediators. Western blot and immunocytochemistry analyses of L1236 cells demonstrated that 15-lipoxygenase-1 was present mainly in the cytosol and that the enzyme translocated to the membrane upon calcium challenge. By immunohistochemistry of Hodgkin lymphoma tumour tissue, 15-lipoxygenase-1 was found to be expressed in primary Hodgkin Reed-Sternberg cells in 17 of 20 (85%) investigated biopsies. The enzyme 15-lipoxygenase-1, however, was not expressed in any of 10 biopsies representing nine different subtypes of non-Hodgkin lymphoma. In essence, the expression of 15-lipoxygenase-1 and the putative formation of eoxins by Hodgkin Reed-Sternberg cells in vivo are likely to contribute to the inflammatory features of Hodgkin lymphoma. These findings may have important diagnostic and therapeutic implications in Hodgkin lymphoma. Furthermore, the discovery of the high 15-lipoxygenase-1 activity in L1236 cells demonstrates that this cell line comprises a useful model system to study the chemical and biological roles of 15-lipoxygenase-1.

In approximately 50% of patients with Hodgkin's lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

The International Prognostic Score (IPS) identifies seven independent factors predicting progression-free and overall survival in advanced stage Hodgkin's disease (HD). The IPS is also applicable in limited disease. However, the IPS does not identify patients with a very poor prognosis. The aim of this study was to define biological markers which may add to the IPS in predicting outcome.One hundred forty-five patients (15 years) with HD of all stages and histopathology subgroups were included. In addition to factors included in the IPS, serum levels of CRP, sCD4, sCD8, sCD25, sCD30, sCD54, interleukin (IL)-10, beta2-microglobulin and thymidine kinase were analysed.The strongest predictors of a poor cause-specific survival (CSS) in univariate analyses were: increased serum levels of IL-10, sCD30 and CRP, anaemia, low levels of albumin (P0.001); stage IV (P = 0.003), ageor = 45 years (P = 0.006), increased serum levels of sCD25 (P = 0.010), low lymphocyte counts (P = 0.020). Serum IL-10 added prognostic information to that achieved by the IPS: patients with a high score and increased serum IL-10 had a very poor outcome with a five-year CSS of 38%. Patients with increased serum levels of sCD30 and a high score also had a poor outcome with a five-year CSS of 54%.Serum levels of IL-10 and sCD30 may add to IPS in prediction of outcome in HD, and should be validated in large, prospective studies.

Epstein–Barr virus (EBV) expression was investigated by immunohistochemistry (latent membrane protein 1 [LMP-1]) and in situ hybridization (EBV encoded RNA [EBER]) in biopsies from 95 patients with untreated Hodgkin’s disease (HD). Tumour EBV status was related to EBV antibody titres, spontaneous and concanavallin A induced blood lymphocyte DNA synthesis, serum levels of soluble (s) CD4, sCD8, sCD25, sCD30, sCD54, β2-microglobulin, thymidine-kinase, routine chemistry, patient characteristics, complete remission and survival. The median follow-up time was 145 months (range 60–257). Tumour EBV-positive (n = 30; 33%) and negative (n = 62; 67%) patients did not differ with regard to sex, age, stage, presence of bulky disease or B-symptoms, remission rate or survival. The proportion of EBV+ cases was significantly higher among patients with mixed cellularity histopathology (58%) as compared to the nodular sclerosis subtype (18%; P < 0.001). The total white blood cell (WBC) counts were significantly lower in EBV+ patients (P < 0.01), who also had significantly higher levels of sCD54 (P < 0.02) and a tendency towards lower levels of sCD30 (P = 0.056). Patients in the tumour EBV+ group had significantly higher IgG antibody titres to restricted early antigen (EA-R) (P < 0.02). Hence, clinical features and outcome were not related to tumour EBV status. However, HD patients with EBV+ tumours had elevated sCD54 levels, higher antibody titres to EA-R and decreased total WBC counts. A potential causal relationship between EBV tumour status and these findings needs to be further explored. © 1999 Cancer Research Campaign

To the editor: First-degree relatives of patients with Hodgkin lymphoma (HL) have a threefold to fourfold increased risk of developing HL, which is stronger when the proband is

Abstract 2491 While much has been discovered in the laboratory regarding the mechanisms of multidrug resistance (MDR), based on phase III clinical trials of ATP-binding cassette (ABC) transporter modulators, these findings have not resulted in appreciable clinical benefit in acute myeloid leukemia (AML) patients. In this pilot study, we investigated the clinical relevance of 380 genes related to MDR in 31 samples from AML patients who had undergone conventional treatment, with 11 patients contributing samples at diagnosis and relapse, using TaqMan Low Density Arrays (TLDA). We and others have shown that the TaqMan-based qRT-PCR is superior to microarrays and SYBR-green-based qRT-PCR because of higher sensitivity and specificity in determining expression of highly homologous gene family members, such as the ABC transporters. 380 genes involved in metabolism, signal transduction, DNA repair, stress response, tumor suppressor activity, oncogenic transformation, apoptosis, and drug uptake or efflux were curated based on their potential role in MDR in the current literature. We found expression of four genes previously unreported in AML in both diagnostic and relapsed patient samples to correlate with duration of complete remission (CR) of induction chemotherapy: SLC7A8 and HSPE1 correlate negatively while GBP1 and ABCD2 correlate positively with CR duration (>2-fold change relative to the median, p2-fold change relative to the median, p2 fold change compared to diagnosis, p2 fold change compared to diagnosis, p Disclosures: No relevant conflicts of interest to declare.

Abstract 205 Background: Little progress in terms of improving survival in patients with chronic myeloid leukemia (CML) was made until the introduction of interferon alpha and allogeneic stem cell transplantation for selected patients in the 1980s. The management changed dramatically with the development of imatinib mesylate, the first tyrosine kinase inhibitor (TKI) that targets the BCR-ABL1 oncoprotein. In Sweden clinical trials started in December 2000 and the drug was approved for clinical use in November 2001. This study evaluates the impact of treatment developments in CML by studying temporal trends in short-term and long-term excess mortality in a population-based setting. Materials and Methods: Using data from the nationwide, population-based Swedish Cancer Registry and Swedish population life-tables stratified by age, sex, and calendar time we characterized trends in relative survival for all patients diagnosed with CML in Sweden 1973–2008 (n=3,173; 1,796 men and 1,377 women; median age 62 years). Patients were categorized into five age groups (79 years) and five calendar periods (1973-1979, 1980–1986, 1987–1993, 1994–2000 and 2001–2008). Six hundred and nine stem cell transplants (539 allogeneic and 70 autologous) were reported to the EBMT registry during the study period. Results: Incidence remained stable over time with a consistent male predominance. Relative survival improved with calendar period with the greatest improvement in the last two calendar periods (figure). Five-year cumulative relative survival ratios (RSRs; 95% confidence intervals) were 0.21 (0.17-0.24), 0.23 (0.20-0.27), 0.37 (0.33-0.41), 0.54 (0.50-0.58) and 0.80 (0.75-0.83) in the five calendar periods, respectively. Ten-year RSRs were 0.06 (0.04-0.08) and 0.78 (0.73-0.83) in the first and last calendar periods, respectively. This improvement was confined to age groups up to 79 years of age but most pronounced in patients below 60 years. The 5-year RSRs for patients diagnosed 2001–2008 were 0.91 (0.85-0.94), 0.87 (0.78-0.92), 0.82 (0.72-0.90), 0.75 (0.61-0.86), and 0.25 (0.10-0.47) for the five age groups, respectively. Older age at diagnosis and male sex were associated with significantly higher excess mortality rates in models adjusted for potential confounding factors. Conclusion: In this large population-based study including > 3,000 CML patients survival increased significantly after 2001 (when imatinib mesylate was approved for clinical use in Sweden) for patients up to 79 years of age. Future studies are needed to assess if very old (>79 years) CML patients may benefit from an increased use of TKIs. Also newly introduced, targeted treatment options for CML need to be evaluated in future population-based studies. Disclosures: No relevant conflicts of interest to declare.

Arachidonic acid metabolites, like prostaglandins and leukotrienes, have been suggested to play an important role not only in inflammation but also in carcinogenesis. We have recently reported that the cysteinyl leukotriene receptor 1 (CysLT1) and 15-lipoxygenase-1 (15-LO-1), a sister enzyme to 5-LO (which is essential for leukotriene synthesis), are expressed by the malignant Hodgkin Reed-Sternberg cells of Hodgkin lymphoma (HL) and certain HL-derived cell lines, and that these cells convert arachidonic acid to the novel pro-inflammatory eoxins (Int J Cancer, 2008; FEBS J, 2008; PNAS, 2008). Given the pleiotropic effects of the CysLT1 receptor and 15-LO-1 and the fact that their expression is strictly regulated in a cell-type and tissue-specific manner, it was of interest to elucidate whether these proteins are exclusively expressed by the tumor cells of the classical HL entities. Therefore, the expression of these molecules was investigated by immunohistochemistry in a broad range of lymphomas (lymphocyte predominance (LP) HL, MALT lymphoma, T cell-derived anaplastic large cell lymphomas (ALCL), immunocytoma, mantle cell lymphoma, B-CLL, Burkitt lymphoma, follicular lymphoma, DLBCL, PTCL, primary mediastinal B cell lymphoma (PMBCL), T cell-rich B cell lymphoma; n=58). The tumor cells in one of four T cell-derived ALCLs, in contrast to all other studied non-Hodgkin lymphomas (NHLs) and LP HL tumors, strongly expressed 15-LO-1. PMBCL was the only NHL entity showing tumor cells positive for the CysLT1 receptor (9 of 10 tumors), and the PMBCL cell line Med-B1 expressed functional CysLT1 receptors, responding with a robust calcium signal upon cysteinyl leukotriene challenge. These findings further corroborate the pathologic overlap between PMBCL and classical HL. Although most cases of PMBCL are identified by routine diagnostics, the distinction between PMBCL and DLBCL may be challenging. After future careful validation in extended patient series, the CysLT1 receptor may be a potential additional marker to phenotypically distinguish these two lymphoma entities.

Lipoxygenases (LO) are a family of structurally related enzymes which catalyze the conversion of the fatty acid arachidonic acid to biologically active metabolites. The key enzyme in leukotriene synthesis, 5- LO, catalyzes the first step in the metabolism of arachidonic acid to leukotriene (LT) C4. This compound and its metabolites LTD4 and LTE4, collectively called cysteinyl leukotrienes (cysLT), can bind to two high-affinity receptors, named cysLT1 and cysLT2. A sister enzyme to 5-LO is 15-LO-1 which also can catalyze the formation of bioactive metabolites. Arachidonic acid metabolites have traditionally been linked to inflammation and asthma but several studies also indicate a role of these metabolites in carcinogenesis In our studies of arachidonic acid metabolism in human lymphomas, we have identified the expression of 15-LO-1 and receptors for cysLT in primary as well as cultured Hodgkin-Reed Sternberg (H-RS) cells. In tumor tissue, H-RS cells positive for 15-LO-1 and the cysLT1 receptor were detected immunohistochemically in 13/15 and 12/16 cases, respectively. The presence of mRNA for 15-LO-1 and the cysLT1 and 2 receptors was confirmed by microarray analysis of laser dissected H-RS cells. Studies of 15-LO-1 gene transcription revealed that STAT6 activation and chromatin remodeling by DNA demethylation and histone acetylation are crucial for transcriptional activation of this gene in cultured H-RS cells. Incubation of the Hodgkin lymphoma-derived cell lines KMH2 and L1236 with LTD4 led to a robust calcium response that was completely abolished in presence of the cysLT1 receptor inhibitor zafirlukast. In L1236 cells, LTD4 induced increased DNA synthesis, interleukin-13 transcription and release of TNF-alpha, interleukin-6 and interleukin-8 in a dose-dependent manner, all of which were inhibited by zafirlukast. Thus, as the H-RS cells lack the expression of 5-LO, we hypothesize that that tumor-associated cysLT-producing inflammatory cells, such as eosinophils and mast cells, contribute to the pathogenesis of Hodgkin lymphoma through induction of tumor cell proliferation and cytokine release. Furthermore, immunohistochemical studies of 20 non-Hodgkin lymphomas revealed the expression of cysLT1 receptors also in primary mediastinal B cell lymphomas (PMBCL), and the functionality of these receptors was confirmed in the PMBCL-derived cell line MedB1. Taken together, these novel findings support a role for arachidonic acid metabolites in the pathogenesis of lymphoma.

Signal transducer and activator of transcription 6 (STAT6) plays a central role in interleukin (IL)-4 and -13 signaling. Upon binding of the cognate receptors by these cytokines, STAT6 becomes phosphorylated by Jak family kinases and subsequently translocates to the nucleus where transcription of its target genes is regulated. Expression of IL-13 and its receptor are common features of Hodgkin lymphoma (HL) tumor cells, the so-called Hodgkin Reed-Sternberg (H-RS) cells, in which this cytokine has been shown to act as an autocrine growth factor. Consequently, constitutively phosphorylated STAT6 with a nuclear localization is a common and distinctive feature of H-RS cells in classical HL. We knocked down STAT6 expression in the HL cell line L1236 with small interfering RNA (siRNA) and found that inhibition of STAT6 activity results in cell growth inhibition, decreased viability and increased apoptosis. The results depict a central role of STAT6 in the growth of H-RS cells and indicate that STAT6 could be a potential target for therapeutic intervention in HL. Moreover, in order to identify the target genes of this transcription factor in H-RS cells, a combined approach of RNA interference and microarray is performed and the results will be presented.

Background: Classical Hodgkin Lymphoma (cHL) is characterized by a minority of malignant cells, the so-called Hodgkin- and Reed Sternberg (H-RS) cells, surrounded by inflammatory cells such as eosinophils, macrophages and mast cells. In contrast to other tumors, the malignant H-RS cells constitute only a few percent of the total cells in the affected tissue. Therefore, it is generally believed that various compounds released by H-RS cells and the interaction between H-RS cells and by- stander cells are of great importance in the pathophysiology of cHL disease. Aim: To characterize the expression and function of cysteinyl leukotriene receptors (CysLTR) in cHL. Methods and results: We have identified functional CysLT1R in a HL cell line as shown by increased intracellular calcium release upon leukotriene (LT) D4 stimulation (100–500 nM). This response was completely blocked after addition of zafirlukast, a specific CysLT1R antagonist. Immunohistochemical studies of paraffin embedded cHL tissue showed H-RS cells positive for CysLT1R in 12 of 16 cHL tumors. Microarray analysis of laser captured H-RS cells from 3 tumors not only confirmed the expression of CysLT1R transcripts, but also showed expression of the CysLT2R gene (P. Murray, personal communication). The possible role of the CysLT2R in H-RS cells will be discussed. The HL cell line was cultured in the presence of LTD4 (100 nM) to investigate the effects of CysLT signaling in H-RS cells. Real-time RT-PCR analysis showed up-regulation of TNF-α, interleukin (IL)-6, IL-8 and IL-13 mRNA after stimulation with LTD4. Furthermore, the effects of LTD4 on cytokine protein secretion by the HL cells were studied by flow cytometry. The results showed a markedly increased secretion of TNF-α, IL-6 and IL-8 upon LTD4 stimulation. In addition, LTD4 stimulated cell proliferation in a dose-dependent manner. Conclusion: Since H-RS cells are surrounded by CysLT producing cells (eosinophils, macrophages and mast cells), these results indicate that CysLT signaling could be of importance in the pathogenesis of cHL by contributing to proliferation of the tumor cell population and the disturbed cytokine features of this tumor. This study was supported by the Swedish Cancer Society.