Greaves P, Clear A, Coutinho R, et al.
Expression of FOXP3, CD68, and CD20 at diagnosis in the microenvironment of classical Hodgkin lymphoma is predictive of outcome.
J Clin Oncol. 2013; 31(2):256-62 [PubMed]

PURPOSE: The immune microenvironment is key to the pathophysiology of classical Hodgkin lymphoma (CHL). Twenty percent of patients experience failure of their initial treatment, and others receive excessively toxic treatment. Prognostic scores and biomarkers have yet to influence outcomes significantly. Previous biomarker studies have been limited by the extent of tissue analyzed, statistical inconsistencies, and failure to validate findings. We aimed to overcome these limitations by validating recently identified microenvironment biomarkers (CD68, FOXP3, and CD20) in a new patient cohort with a greater extent of tissue and by using rigorous statistical methodology.
PATIENTS AND METHODS: Diagnostic tissue from 122 patients with CHL was microarrayed and stained, and positive cells were counted across 10 to 20 high-powered fields per patient by using an automated system. Two statistical analyses were performed: a categorical analysis with test/validation set-defined cut points and Kaplan-Meier estimated outcome measures of 5-year overall survival (OS), disease-specific survival (DSS), and freedom from first-line treatment failure (FFTF) and an independent multivariate analysis of absolute uncategorized counts.
RESULTS: Increased CD20 expression confers superior OS. Increased FOXP3 expression confers superior OS, and increased CD68 confers inferior FFTF and OS. FOXP3 varies independently of CD68 expression and retains significance when analyzed as a continuous variable in multivariate analysis. A simple score combining FOXP3 and CD68 discriminates three groups: FFTF 93%, 62%, and 47% (P < .001), DSS 93%, 82%, and 63% (P = .03), and OS 93%, 82%, and 59% (P = .002).
CONCLUSION: We have independently validated CD68, FOXP3, and CD20 as prognostic biomarkers in CHL, and we demonstrate, to the best of our knowledge for the first time, that combining FOXP3 and CD68 may further improve prognostic stratification.

Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London

Montoto S, Shaw K, Okosun J, et al.
HIV status does not influence outcome in patients with classical Hodgkin lymphoma treated with chemotherapy using doxorubicin, bleomycin, vinblastine, and dacarbazine in the highly active antiretroviral therapy era.
J Clin Oncol. 2012; 30(33):4111-6 [PubMed]

PURPOSE: The prognosis of HIV-infected patients with non-Hodgkin lymphoma in the highly active antiretroviral therapy (HAART) era approaches that of the general population when they are treated with the same protocols. We analyzed the outcome of patients with Hodgkin lymphoma (HL) treated with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) in the HAART era according to HIV serostatus to establish whether this also holds true for HL.
PATIENTS AND METHODS: From 1997 to 2010, 224 patients newly diagnosed with HL, of whom 93 were HIV positive, were consecutively treated with ABVD chemotherapy. HIV-positive patients had more high-risk disease according to the International Prognostic Score (IPS) than HIV-negative patients (IPS≥3: 68% v 26%, respectively; P<.001). Forty-seven HIV-positive patients had a CD4 count less than 200/μL, and 92 patients received HAART during chemotherapy.
RESULTS: The complete response rate was 74% for HIV-positive patients and 79% for HIV-negative patients (P=not significant). After a median follow-up of 60 months (range, 8 to 174 months), 23 patients (16 HIV-negative and seven HIV-positive patients) have experienced relapse at a median time of 6 months (range, 1 to 106 months). Five-year event-free survival (EFS) was 59% (95% CI, 47% to 70%) for HIV-positive patients and 66% (95% CI, 57% to 74%) for HIV-negative patients (P=not significant). Five-year overall survival (OS) was 81% (95% CI, 69% to 89%) and 88% (95% CI, 80% to 93%) for HIV-positive and HIV-negative patients, respectively (P=not significant). HIV status did not predict OS or EFS on multivariate analysis including IPS and HIV status.
CONCLUSION: This mature study demonstrates that HIV-positive patients with HL have more extensive disease with more adverse prognostic factors than HIV-negative patients, but when treated with ABVD, HIV infection does not adversely affect OS or EFS.

Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, Chelsea and Westminster Hospital, 45 Little Britain, EC1A 7BE, London

Yeoh KW, Mikhaeel NG
Are we ready for positron emission tomography/computed tomography-based target volume definition in lymphoma radiation therapy?
Int J Radiat Oncol Biol Phys. 2013; 85(1):14-20 [PubMed]

Fluorine-18 fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) has become indispensable for the clinical management of lymphomas. With consistent evidence that it is more accurate than anatomic imaging in the staging and response assessment of many lymphoma subtypes, its utility continues to increase. There have therefore been efforts to incorporate PET/CT data into radiation therapy decision making and in the planning process. Further, there have also been studies investigating target volume definition for radiation therapy using PET/CT data. This article will critically review the literature and ongoing studies on the above topics, examining the value and methods of adding PET/CT data to the radiation therapy treatment algorithm. We will also discuss the various challenges and the areas where more evidence is required.

Department of Clinical Oncology, Guy's and St. Thomas' Hospital, London

Proctor SJ, Wilkinson J, Jones G, et al.
Evaluation of treatment outcome in 175 patients with Hodgkin lymphoma aged 60 years or over: the SHIELD study.
Blood. 2012; 119(25):6005-15 [PubMed]

The SHIELD program for Hodgkin lymphoma in patients 60 years of age or older, prospectively evaluated clinical features and outcome in a large patient cohort (n = 175). The central element was a phase 2 study of VEPEMB chemotherapy (n = 103, median age 73 years) incorporating comorbidity assessment. A total of 72 other patients were treated off-study but registered prospectively and treated concurrently with: ABVD (n = 35); CLVPP (n = 19), or other (n = 18). Of VEPEMB patients, 31 had early-stage disease (stage 1A/2A) and received VEPEMB 3 times plus radiotherapy. Median follow-up was 36 months. Complete remission (CR) rate (intention-to-treat) was 74% and 3-year overall survival (OS) and progression-free survival (PFS) were 81% and 74%, respectively. A total of 72 patients had advanced-stage disease (stage 1B/2B/3 or 4) and received VEPEMB 6 times. CR rate was 61% with 3-year OS and PFS of 66% and 58%, respectively. Of patients achieving CR, 13% with early-stage and 5% with advanced-stage disease progressed. Overall treatment-related mortality was 7%. In patients treated with curative intent with VEPEMB, ABVD, and CLVPP (n = 157), CR linked to several factors in univariate analysis. In a Cox regression model only, obtaining CR remained significant for OS and CR plus comorbidity and age for PFS. RS-EBV status had no significant effect on outcome.

Academic Haematology, Northern Institute of Cancer Research, Newcastle University, Newcastle upon Tyne

Simpson AA, Mohammed F, Salim M, et al.
Structural and energetic evidence for highly peptide-specific tumor antigen targeting via allo-MHC restriction.
Proc Natl Acad Sci U S A. 2011; 108(52):21176-81 [PubMed] Free Access to Full Article

Immunotherapies targeting peptides presented by allogeneic MHC molecules offer the prospect of circumventing tolerance to key tumor-associated self-antigens. However, the degree of antigen specificity mediated by alloreactive T cells, and their ability to discriminate normal tissues from transformed cells presenting elevated antigen levels, is poorly understood. We examined allorecognition of an HLA-A2-restricted Hodgkin's lymphoma-associated antigen and were able to isolate functionally antigen-specific allo-HLA-A2-restricted T cells from multiple donors. Binding and structural studies, focused on a prototypic allo-HLA-A2-restricted T-cell receptor (TCR) termed NB20 derived from an HLA-A3 homozygote, suggested highly peptide-specific allorecognition that was energetically focused on antigen, involving direct recognition of a distinct allopeptide presented within a conserved MHC recognition surface. Although NB20/HLA-A2 affinity was unremarkable, TCR/MHC complexes were very short-lived, consistent with suboptimal TCR triggering and tolerance to low antigen levels. These data provide strong molecular evidence that within the functionally heterogeneous alloreactive repertoire, there is the potential for highly antigen-specific "allo-MHC-restricted" recognition and suggest a kinetic mechanism whereby allo-MHC-restricted T cells may discriminate normal from transformed tissue, thereby outlining a suitable basis for broad-based therapeutic targeting of tolerizing tumor antigens.

Birmingham Cancer Research UK Cancer Centre, School of Cancer Sciences, University of Birmingham, Birmingham B15 2TT

Ma YP, van Leeuwen FE, Cooke R, et al.
FGFR2 genotype and risk of radiation-associated breast cancer in Hodgkin lymphoma.
Blood. 2012; 119(4):1029-31 [PubMed]

Women treated at young ages with supradiaphragmatic radiotherapy for Hodgkin lymphoma (HL) have a highly increased risk of breast cancer. For personalized advice and follow-up regimens for patients, information is needed on how the radiotherapy-related risk is affected by other breast cancer risk factors. Genome-wide association studies have identified 14 independently replicated common single nucleotide polymorphisms that influence breast cancer risk. To examine whether these variants contribute to risk of radiation-associated breast cancer in HL, we analyzed 2 independent case-control series, from the United Kingdom and The Netherlands, totaling 693 HL patients, 232 with breast cancer and 461 without. rs1219648, which annotates the FGFR2 gene, was associated with risk in both series (combined per-allele odds ratio = 1.59, 95% confidence interval: 1.26-2.02; P = .000111). These data provide evidence that genetic variation in FGFR2 influences radiation-induced breast cancer risk.

Section of Cancer Genetics, Institute of Cancer Research, 15 Cotswold Rd., Sutton, Surrey

Swerdlow AJ, Higgins CD, Smith P, et al.
Second cancer risk after chemotherapy for Hodgkin's lymphoma: a collaborative British cohort study.
J Clin Oncol. 2011; 29(31):4096-104 [PubMed]

PURPOSE: We investigated the long-term risk of second primary malignancy after chemotherapy for Hodgkin's lymphoma (HL) in a much larger cohort than any yet published, to our knowledge.
PATIENTS AND METHODS: We followed 5,798 patients with HL treated with chemotherapy in Britain from 1963 to 2001--of whom 3,432 also received radiotherapy--to assess second primary malignancy risks compared with general population-based expectations.
RESULTS: Second malignancies occurred in 459 cohort members. Relative risk (RR) of second cancer was raised after chemotherapy alone (RR, 2.0; 95% CI, 1.7 to 2.4) but was much lower than after combined modalities (RR, 3.9; 95% CI, 3.5 to 4.4). After chemotherapy alone, there were significantly raised risks of lung cancer, non-HL, and leukemia, each contributing approximately equal absolute excess risk. After combined modalities, there were raised risks of these and several other cancers. Second cancer risk peaked 5 to 9 years after chemotherapy alone, but it remained raised for 25 years and longer after combined modalities. Risk was raised after each common chemotherapy regimen except, based on limited numbers and follow-up, adriamycin, bleomycin, vinblastine, and dacarbazine. The age and time-course relations of lung cancer differed between chemotherapy alone and combined modalities.
CONCLUSION: Although chemotherapy alone leads to raised risk of second malignancy, this risk is lower and affects fewer anatomic sites than that after combined modalities, and it is slight if at all after 15 years follow-up. The mechanism of lung cancer etiology may differ between chemotherapy and radiotherapy.

Section of Epidemiology, Sir Richard Doll Building, Institute of Cancer Research, Sutton, Surrey SM2 5NG

Leonard S, Wei W, Anderton J, et al.
Epigenetic and transcriptional changes which follow Epstein-Barr virus infection of germinal center B cells and their relevance to the pathogenesis of Hodgkin's lymphoma.
J Virol. 2011; 85(18):9568-77 [PubMed] Free Access to Full Article

Although Epstein-Barr virus (EBV) usually establishes an asymptomatic lifelong infection, it is also implicated in the development of germinal center (GC) B-cell-derived malignancies, including Hodgkin's lymphoma (HL). Following primary infection, EBV remains latent in the memory B-cell population, where host-driven methylation of viral DNA contributes to the repression of viral gene expression. However, it is still unclear how EBV harnesses the cell's methylation machinery in B cells, how this contributes to viral persistence, and what impact this has on the methylation of cellular genes. We show that EBV infection of GC B cells is followed by upregulation of the DNA methyltransferase DNMT3A and downregulation of DNMT3B and DNMT1. We show that the EBV latent membrane protein 1 (LMP1) oncogene downregulates DNMT1 and that DNMT3A binds to the viral promoter Wp. Genome-wide promoter arrays performed with these cells showed that EBV-associated methylation changes in cellular genes were not randomly distributed across the genome but clustered at chromosomal locations, consistent with an instructive pattern of methylation, and were in part determined by promoter CpG content. Both DNMT3B and DNMT1 were downregulated and DNMT3A was upregulated in HL cell lines, recapitulating the pattern of expression observed following EBV infection of GC B cells. We also found, by using gene expression profiling, that genes differentially expressed following EBV infection of GC B cells were significantly enriched for those reported to be differentially expressed in HL. These observations suggest that EBV-infected GC B cells are a useful model for studying virus-associated changes contributing to the pathogenesis of HL.

College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK B15 2TT

Moutsianas L, Enciso-Mora V, Ma YP, et al.
Multiple Hodgkin lymphoma-associated loci within the HLA region at chromosome 6p21.3.
Blood. 2011; 118(3):670-4 [PubMed]

Since an association between the human leukocyte antigen (HLA) region and Hodgkin lymphoma (HL) was first reported in 1967, many studies have reported associations between HL risk and both single nucleotide polymorphism (SNP) and classic HLA allele variation in the major histocompatibility complex. However, population stratification and the extent and complexity of linkage disequilibrium within the major histocompatibility complex have hindered efforts to fine-map causal signals. Using SNP data to impute alleles at classic HLA loci, we have conducted an integrated analysis of HL risk within the HLA region in 582 early-onset HL cases and 4736 controls. We confirm that the strongest signal of association comes from an SNP located in the class II region, rs6903608 (odds ratio [OR] = 1.79, P = 6.63 × 10(-19)), which is unlikely to be driven by association to HLA-DRB, DQA, or DQB alleles. In addition, we identify independent signals at rs2281389 (OR = 1.73, P = 6.31 × 10(-13)), a SNP that maps closely to HLA-DPB1, and the class II HLA allele DQA1*02:01 (OR = 0.56, P = 1.51 × 10(-7)). These data suggest that multiple independent loci within the HLA class II region contribute to the risk of developing early-onset HL.

Department of Statistics, University of Oxford, Oxford

Peggs KS, Kayani I, Edwards N, et al.
Donor lymphocyte infusions modulate relapse risk in mixed chimeras and induce durable salvage in relapsed patients after T-cell-depleted allogeneic transplantation for Hodgkin's lymphoma.
J Clin Oncol. 2011; 29(8):971-8 [PubMed]

PURPOSE: Reduced-intensity conditioning has minimized nonrelapse-related mortality rates after allogeneic transplantation in patients with Hodgkin's lymphoma, and relapse has now become the major cause for treatment failure. We aimed to assess the impact of donor lymphocyte infusions (DLIs) on relapse incidence when administered for mixed chimerism and their utility as salvage therapy when given for relapse.
PATIENTS AND METHODS: This study reports the outcomes of 76 consecutive patients with multiply relapsed or refractory Hodgkin's lymphoma who underwent allogeneic transplantation that incorporated in vivo T-cell depletion. Forty-two patients had related donors and 34 had unrelated donors. DLIs were administered in a dose-escalating fashion to 22 patients for mixed chimerism (median time of first dose, 9 months post-transplantation) and to 24 patients for relapse.
RESULTS: Three-year donor lymphocyte-related mortality was 7%, relating mainly to the induction of graft-versus-host disease. Nineteen (86%) of 22 patients receiving donor lymphocytes for mixed chimerism converted to full donor status. Four-year relapse incidence was 5% in these 22 patients compared with 43% in patients who remained relapse free but full donor chimeras at 9 months post-transplantation (P = .0071). Nineteen (79%) of 24 patients receiving donor lymphocytes for relapse responded (14 complete responses, five partial responses). Four-year overall survival from relapse was 59% in recipients of donor lymphocytes, contributing to a 4-year overall survival from transplantation of 64% and a 4-year current progression-free survival of 59% in all 76 patients.
CONCLUSION: These data demonstrate the potential for allogeneic immunotherapy with donor lymphocytes both to reduce relapse risk and to induce durable antitumor responses in patients with Hodgkin's lymphoma after hematopoietic stem-cell transplantation that incorporates in vivo T-cell depletion.

Department of Haematology, UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley St, London WC1E 6BT United Kingdom. karl.peggs@cancer.ucl.ac.uk

Johnson PW, Sydes MR, Hancock BW, et al.
Consolidation radiotherapy in patients with advanced Hodgkin's lymphoma: survival data from the UKLG LY09 randomized controlled trial (ISRCTN97144519).
J Clin Oncol. 2010; 28(20):3352-9 [PubMed]

PURPOSE: This study analyzed the outcomes of nonrandomized consolidation radiotherapy (RT) given after chemotherapy in the initial treatment of advanced Hodgkin's lymphoma (HL). The results were collected prospectively within a randomized controlled trial of induction chemotherapy.
PATIENTS AND METHODS: Patients were randomly assigned between doxorubicin, bleomycin, vinblastine, and dacarbazine and one of two prespecified multidrug regimens. At least six cycles of chemotherapy were planned, with up to eight for patients showing slower response. Involved-field RT was recommended for incomplete response to chemotherapy or bulk disease at presentation. The primary outcome measure was progression-free survival (PFS), landmarked from the end of chemotherapy.
RESULTS: Among 807 patients randomly assigned, 702 achieved objective response. Postchemotherapy RT for consolidation was reported in 300 (43%). With median follow-up of 6.9 years, 161 PFS events and 83 deaths were reported. Baseline characteristics showed more patients with bulk disease having RT (190 [63%] v 111 [28%]) and only partial response after chemotherapy (150 [50%] v 36 [9%]). Other baseline characteristics were similar. PFS was superior for patients having RT (hazard ratio [HR], 0.43; 95% CI, 0.30 to 0.60) with 5-year PFS 71% without RT, 86% with RT. A similar advantage was seen for overall survival (HR, 0.47; 95% CI, 0.29 to 0.77). There was no evidence of heterogeneity of treatment effect across subgroups.
CONCLUSION: Patients who received consolidation RT apparently had better outcomes, consistently across all prognostic groups which persisted in multivariate analysis. This suggests that RT contributes significantly to the cure rate for advanced HL, although patient selection for combined modality treatment requires better definition in prospective trials.

University of Southampton, Medical Research Council (MRC) Clinical Trials Unit, London

Grinyer A
The late effects of mantle field radiotherapy: the information and support needs of women survivors of Hodgkin's disease.
Eur J Oncol Nurs. 2010; 14(3):183-9 [PubMed]

PURPOSE: To improve understanding of the information and support needs of women at risk from breast cancer after earlier treatment with mantle field radio therapy for Hodgkin's disease.
METHOD: A multiple case study approach presenting the detailed accounts of three participants has been used to represent the issues raised by a wider sub-set of 15 Hodgkin's disease survivors. These women were participants in a larger qualitative study of 50 long term survivors of adolescent cancer. They were interviewed in depth either face to face or via the telephone. Data were analysed using methods of data reduction, display and conclusion drawing, three case studies were then purposively selected to illustrate key themes in the wider data set.
KEY RESULTS: Discovering the risk of late effect breast cancer, particularly via the media, caused shock. Reliable information was difficult to locate and the support needs of the women were not met by medical services. The impact of the new risk was exacerbated by the earlier cancer experience.
CONCLUSION: Information on the risk of late effects to cancer survivors regarded as trustworthy is not easy to access. When the risk of a treatment related malignancy is identified there must be a reliable way of informing both those at risk and primary health care physicians who then need to take into account the cancer history of the patient and its ongoing effect on their emotional vulnerability.

Division of Health Research, School of Health and Medicine, Lancaster University, Lancaster, LA1 4YT

Hoskin PJ, Lowry L, Horwich A, et al.
Randomized comparison of the stanford V regimen and ABVD in the treatment of advanced Hodgkin's Lymphoma: United Kingdom National Cancer Research Institute Lymphoma Group Study ISRCTN 64141244.
J Clin Oncol. 2009; 27(32):5390-6 [PubMed]

PURPOSE: This multicenter, prospective, randomized controlled trial compared the efficacy and toxicity of two chemotherapy regimens in advanced Hodgkin's lymphoma (HL): the weekly alternating Stanford V and the standard, twice-weekly regimen of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD).
PATIENTS AND METHODS: Patients had stage IIB, III, or IV disease or had stages I to IIA disease with bulky disease or other adverse features. Radiotherapy was administered in both arms to sites of previous bulk (> 5 cm) and to splenic deposits, although this was omitted in the latter part of the trial for patients achieving complete remission (CR) in the ABVD arm. A total of 520 patients were randomly assigned and were assessed for the primary outcome measure of progression-free survival (PFS). Five hundred patients received protocol treatment, and radiotherapy was administered to 73% in the Stanford V arm and to 53% in the ABVD arm.
RESULTS: The overall response rates after completion of all treatment were 91% for Stanford V and 92% for ABVD. During a median follow-up of 4.3 years, there was no evidence of a difference in projected 5-year PFS and overall survival (OS) rates (76% and 90%, respectively, for ABVD; 74% and 92%, respectively, for Stanford V). More pulmonary toxicity was reported for ABVD, whereas other toxicities were more frequent with Stanford V.
CONCLUSION: In a large, randomized trial, the efficacies of Stanford V and ABVD were comparable when given in combination with appropriate radiotherapy.

Mount Vernon Cancer Centre, Mount Vernon Hospital, Rickmansworth Rd, Northwood, Middlesex HA6 2RN

Lake A, Shield LA, Cordano P, et al.
Mutations of NFKBIA, encoding IkappaB alpha, are a recurrent finding in classical Hodgkin lymphoma but are not a unifying feature of non-EBV-associated cases.
Int J Cancer. 2009; 125(6):1334-42 [PubMed]

A consistent feature of the Hodgkin and Reed-Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) is the constitutive activation of NF-kappaB transcription factors. In Epstein-Barr virus (EBV)-associated cases of cHL, expression of viral antigens most probably leads to NF-kappaB activation but for non-EBV-associated cases, the mechanism is not clear. Previous small studies have demonstrated deleterious mutations of NFKBIA, the gene encoding IkappaB alpha, in HRS cells. In the present study, we aimed to establish the frequency of NFKBIA mutation in cHL by investigating a larger series of cases and to determine whether these mutations are a characteristic feature of non-EBV-associated cHL. Single HRS cells from 20 cases of cHL were analysed by PCRs covering all 6 exons of the gene. Clonal deleterious mutations were detected in 3 cases and in 1 case both alleles of the gene were shown to harbour mutations. NFKBIA mutations were detected only in non-EBV-associated cases but the majority of these cases had wild-type NFKBIA. It remains possible that defects in genes encoding other inhibitors of NF-kappaB, such as TNFAIP3 (A20) and CYLD, are involved in the latter cases, as described for one case in this series.

LRF Virus Centre, Institute of Comparative Medicine, University of Glasgow, Glasgow

Pang D, Alston RD, Eden TO, Birch JM
Cancer risks among relatives of children with Hodgkin and non-Hodgkin lymphoma.
Int J Cancer. 2008; 123(6):1407-10 [PubMed]

A role for genetic susceptibility in the aetiology of childhood lymphomas was investigated in 454 families of children with histologically confirmed Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL) from Northwest England. Cancers in parents were obtained from the UK National Health Service Central Register and in other close relatives by interview with the parents. The cancer incidence among relatives was compared with expected incidence based on cancer registry data for England. There were 197 cancers in relatives (SIR 1.0 95% CI 0.8-1.1). In families of children with HL, there was an excess of HL in the first degree relatives (SIR 5.8 95% CI 1.2-16.9). Excesses of HL diagnosed under population median age (SIR 4.1 95% CI 1.1-10.6) were seen among all relatives and relatives of children who were below the median age at diagnosis (SIR 5.5 95% CI 1.1-16.0). In families of children with NHL, there were non-significant excesses of central nervous system (CNS) tumours in the first degree relatives (SIR 2.9 95% CI 0.8-7.4) and in the second and third degree relatives (SIR 1.5). There were significant excesses of CNS tumours diagnosed under the population median age (SIR 2.8 95% CI 1.1-5.8) in all relatives. Excess CNS tumours were also seen among relatives of children below the median age at diagnosis (SIR 3.2 95% CI 1.1-7.6). In conclusion, genetic susceptibility in some families of children with lymphoma might be operating, but aetiologies in HL and NHL appear to be different. Possible interpretations of our findings, in the context of putative genetic and infectious aetiologies, are discussed.

University of Manchester and Cancer Research UK, Paediatric and Familial Cancer Research Group, Royal Manchester Children's Hospital, Manchester

Baumforth KR, Birgersdotter A, Reynolds GM, et al.
Expression of the Epstein-Barr virus-encoded Epstein-Barr virus nuclear antigen 1 in Hodgkin's lymphoma cells mediates Up-regulation of CCL20 and the migration of regulatory T cells.
Am J Pathol. 2008; 173(1):195-204 [PubMed] Free Access to Full Article

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.

Cancer Research United Kingdom Institute for Cancer Studies, University of Birmingham, Birmingham

Flavell JR, Baumforth KR, Wood VH, et al.
Down-regulation of the TGF-beta target gene, PTPRK, by the Epstein-Barr virus encoded EBNA1 contributes to the growth and survival of Hodgkin lymphoma cells.
Blood. 2008; 111(1):292-301 [PubMed]

The Epstein-Barr virus (EBV) contributes to the growth and survival of Hodgkin lymphoma (HL) cells. Here we report that down-regulation of the transforming growth factor-beta (TGF-beta) target gene, protein tyrosine phosphatase receptor kappa (PTPRK), followed EBV infection of HL cells and was also more frequently observed in the Hodgkin and Reed-Sternberg (HRS) cells of EBV-positive compared with EBV-negative primary HL. The viability and proliferation of EBV-positive HL cells was decreased by overexpression of PTPRK, but increased following the knockdown of PTPRK expression in EBV-negative HL cells, demonstrating that PTPRK is a functional tumor suppressor in HL. EBV suppressed the TGF-beta-mediated activation of PTPRK expression, suggesting disruption of TGF-beta signaling upstream of PTPRK. This was confirmed when we showed that the Epstein-Barr nuclear antigen-1 (EBNA1) decreased Smad2 protein levels and that this was responsible for PTPRK down-regulation. EBNA1 decreased the half-life of Smad2 but did not interact with Smad2. By down-regulating Smad2 protein expression, EBNA1 apparently disables TGF-beta signaling, which subsequently decreases transcription of the PTPRK tumor suppressor. We speculate that loss of the phosphatase function of PTPRK may activate as-yet-unidentified growth-promoting protein tyrosine kinases, which in turn contribute to the pathogenesis of EBV-positive HL.

Cancer Research UK Institute for Cancer Studies, The Medical School, University of Birmingham, Vincent Drive, Edgbaston, Birmingham, B15 2TT

Wilson KS, Freeland JM, Gallagher A, et al.
Measles virus and classical Hodgkin lymphoma: no evidence for a direct association.
Int J Cancer. 2007; 121(2):442-7 [PubMed]

A proportion of Hodgkin lymphoma (HL) cases are causally associated with the Epstein-Barr virus (EBV) but the aetiology of the remaining cases remains obscure. Over the last 3 decades several studies have found an association between HL and measles virus (MV) including a recent cohort study describing the detection of MV antigens in Hodgkin and Reed-Sternberg cells, the tumour cells in HL. In the present study we looked at the relationship between history of MV infection and risk of developing HL in a population-based, case/control study of HL. In addition we used immunohistochemistry and RT-PCR to look for direct evidence of MV in HL biopsies. There was no significant difference in the proportion of cases reporting previous measles compared to controls in the entire data set or when young adults were considered separately. Using a robust immunohistochemical assay for MV infection, we failed to find evidence of MV in biopsies from 97 cases of HL and RT-PCR studies similarly gave negative results. This study therefore provides no evidence that MV is directly involved in the development of HL. However, when age at first reported MV infection was investigated, significant differences emerged with children infected before school-age having higher risk, especially of EBV-ve HL, when compared with children infected at older ages; the interpretation of these latter results is unclear.

LRF Virus Centre, Institute of Comparative Medicine, University of Glasgow, Glasgow

Dutton A, Woodman CB, Chukwuma MB, et al.
Bmi-1 is induced by the Epstein-Barr virus oncogene LMP1 and regulates the expression of viral target genes in Hodgkin lymphoma cells.
Blood. 2007; 109(6):2597-603 [PubMed]

Polycomb group (PcG) proteins are chromatin modifiers that are necessary for the maintenance and renewal of embryonic and adult stem cells. However, overexpression of the PcG protein, Bmi-1, causes lymphoma in transgenic mice. We show that Bmi-1 is up-regulated in Hodgkin lymphoma (HL) cells by the Epstein-Barr virus (EBV) oncogene latent membrane protein-1 (LMP1) and that this up-regulation is mediated by NF-kappaB signaling. We also show that Bmi-1 is up-regulated by NF-kappaB in EBV-negative HL cells. Down-regulation of LMP1 and Bmi-1 decreased the survival of HL cells, suggesting that Bmi-1 may mediate the prosurvival effects of LMP1-induced NF-kappaB signaling in HL cells. Transcriptional targets of Bmi-1 were identified after its knockdown in an HL cell line. We show here that Bmi-1 and LMP1 down-regulate the ataxia telangiectasia-mutated (ATM) tumor suppressor and conclude that Bmi-1 contributes to LMP1-induced oncogenesis in HL.

Cancer Research UK Institute for Cancer Studies, The Medical School, University of Birmingham, Edgbaston

Taylor AJ, Winter DL, Stiller CA, et al.
Risk of breast cancer in female survivors of childhood Hodgkin's disease in Britain: a population-based study.
Int J Cancer. 2007; 120(2):384-91 [PubMed]

There is now widespread evidence that female survivors of Hodgkin's disease who have been treated with supradiaphragmatic radiotherapy are at an increased risk of breast cancer. Mantle irradiation, which includes irradiation of the mediastinum, conveys a particularly high risk. Previously published studies have found a wide variation in risk. To provide British estimates of risk to inform surveillance programmes, we carried out the first British population-based cohort study of breast cancer in female survivors of childhood Hodgkin's disease. From the underlying cohort of the British Childhood Cancer Survivor Study, a cohort of 18,123 British 5-year survivors of childhood cancer diagnosed between 1940 and 1991, there were in total 383 female 5-year survivors of childhood Hodgkin's disease. Sixteen of these 383 survivors went on to develop invasive breast cancer subsequent to 5-year survival (standardised incidence ratio, 11.5; 95% confidence interval (95% CI), 6.6-18.6) and all of these 16 survivors had been treated with supradiaphragmatic irradiation as treatment for childhood Hodgkin's disease. The cumulative risk of breast cancer by 25 years of follow up was 9.9% (95% CI, 3.3-16.6) for all patients and 12.2% (95% CI, 4.3-20.1) for those treated with supradiaphragmatic radiotherapy. The cumulative risk of breast cancer in female survivors of childhood Hodgkin's disease in Britain is at the lower end of previous estimates. We hope that our data may provide a basis for future surveillance and for counselling survivors as to their likely risk of breast cancer.

Centre for Childhood Cancer Survivor Studies, Department of Public Health and Epidemiology, The University of Birmingham, Edgbaston, Birmingham

Johnson PW, Radford JA, Cullen MH, et al.
Comparison of ABVD and alternating or hybrid multidrug regimens for the treatment of advanced Hodgkin's lymphoma: results of the United Kingdom Lymphoma Group LY09 Trial (ISRCTN97144519).
J Clin Oncol. 2005; 23(36):9208-18 [PubMed]

PURPOSE: To perform an open-label, randomized, controlled trial comparing treatment with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) with two multidrug regimens (MDRs) for advanced Hodgkin's lymphoma (HL).
PATIENTS AND METHODS: Eight hundred seven patients with advanced HL (stage III to IV, or earlier stage with systemic symptoms or bulky disease) were randomly assigned between ABVD and MDR specified before randomization as alternating chlorambucil, vinblastine, procarbazine, and prednisolone (ChlVPP) with prednisolone, doxorubicin, bleomycin, vincristine, and etoposide (PABIOE), or hybrid ChlVPP/etoposide, vincristine, and doxorubicin (EVA). Radiotherapy was planned for incomplete response or initial bulk disease.
RESULTS: At 52 months median follow-up, 212 event-free survival (EFS) events (disease progression or any death) were reported. In the primary comparison, at 3 years EFS was 75% (95% CI, 71% to 79%) for ABVD and 75% (95% CI, 70% to 79%) for MDRs (hazard ratio [HR] = 1.05; 95% CI, 0.8 to 1.37; HR more than 1.0 favors ABVD). The 3-year overall survival (OS) rates were 90% (95% CI, 87% to 93%) in patients allocated ABVD and 88% (95% CI, 84% to 91%) in patients allocated MDRs (HR = 1.22; 95% CI, 0.84 to 1.77). Patients receiving MDRs experienced more grade 3/4 infection, mucositis, and neuropathy. One occurrence of myelodysplastic syndrome was reported, but no acute leukemia was reported. When the two MDRs are compared separately with ABVD, neither the alternating nor the hybrid regimen showed a statistically significant difference from ABVD for EFS or OS. Subgroup analysis suggested that MDRs may be associated with poorer outcomes in older patients (heterogeneity test of OS older or younger than 45 years, P = .020).
CONCLUSION: There was no evidence of significant difference in EFS or OS between ABVD and MDRs in the trial overall or if the two MDR versus ABVD comparisons are considered separately. ABVD remains the standard for treatment of advanced HL.

Cancer Research UK Clinical Centre, Somers Cancer Research Building, Southampton General Hospital, Southampton

Jarrett RF, Stark GL, White J, et al.
Impact of tumor Epstein-Barr virus status on presenting features and outcome in age-defined subgroups of patients with classic Hodgkin lymphoma: a population-based study.
Blood. 2005; 106(7):2444-51 [PubMed]

The association between tumor Epstein-Barr virus (EBV) status and clinical outcome in Hodgkin lymphoma (HL) is controversial. This population-based study assessed the impact of EBV status on survival in age-stratified cohorts of adults with classic HL (cHL). Data from 437 cases were analyzed with a median follow-up of 93 months. Overall survival (OS) was significantly better for EBV-negative compared with EBV-positive patients (P < .001), with 5-year survival rates of 81% and 66%, respectively; disease-specific survival (DSS) was also greater for EBV-negative patients (P = .03). The impact of EBV status varied with age at diagnosis. In patients aged 16 to 34 years, EBV-associated cases had a survival advantage compared with EBV-negative cases, but differences were not statistically significant (P = .21). Among patients 50 years or older, EBV positivity was associated with a significantly poorer outcome (P = .003). Excess deaths occurred in EBV-positive patients with both early- and advanced-stage disease. In multivariate analysis of OS in the older patients, EBV status retained statistical significance after adjusting for the effects of sex, stage, and B symptoms (P = .01). Impaired immune status may contribute to the development of EBV-positive cHL in older patients, and strategies aimed at boosting the immune response should be investigated in the treatment of these patients.

Leukaemia Research Fund (LRF) Virus Centre, Institute of Comparative Medicine, University of Glasgow, Glasgow

Baumforth KR, Flavell JR, Reynolds GM, et al.
Induction of autotaxin by the Epstein-Barr virus promotes the growth and survival of Hodgkin lymphoma cells.
Blood. 2005; 106(6):2138-46 [PubMed]

A proportion of patients with Hodgkin lymphoma carry Epstein-Barr virus (EBV), an oncogenic herpesvirus, in their tumor cells. Although it is generally assumed that EBV contributes to the malignant phenotype of Hodgkin lymphoma cells, direct evidence in support of this is lacking. Here we show that EBV infection of Hodgkin lymphoma cells results in the induction of autotaxin, a secreted tumor-associated factor with lysophospholipase-D activity. Up-regulation of autotaxin increased the generation of lysophosphatidic acid (LPA) and led to the enhanced growth and survival of Hodgkin lymphoma cells, whereas specific down-regulation of autotaxin decreased LPA levels and reduced cell growth and viability. In lymphoma tissues, autotaxin expression was mainly restricted to CD30+ anaplastic large-cell lymphomas and Hodgkin lymphoma; in the latter, high levels of autotaxin were strongly associated with EBV positivity (P = .006). Our results identify the induction of autotaxin and the subsequent generation of LPA as key molecular events that mediate the EBV-induced growth and survival of Hodgkin lymphoma cells and suggest that this pathway may provide opportunities for novel therapeutic intervention.

CRUK Institute for Cancer Studies, The Medical School, University of Birmingham, Edgbaston, Birmingham, B15 2TT

Osborne J, Lake A, Alexander FE, et al.
Germline mutations and polymorphisms in the NFKBIA gene in Hodgkin lymphoma.
Int J Cancer. 2005; 116(4):646-51 [PubMed]

Somatic inactivation of NFKBIA, the gene encoding IkappaBalpha, is a frequent occurrence in the malignant Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin lymphoma (HL). Impairment of IkappaBalpha function results in deregulated NF-kappaB activity, a characteristic of HRS cells. The molecular basis for familial HL, which accounts for approximately 4% of all HL cases, is unclear. To date, familial HL cases have not been evaluated for germline NFKBIA mutations. We screened the entire NFKBIA gene in 8 individuals with familial HL but found no mutations in the coding region or promoter sequences. We identified the first germline NFKBIA missense mutation in a patient with presumed sporadic HL. The frequency of 4 polymorphisms within the NFKBIA gene and promoter region was investigated in a series of HL and control samples; no significant differences emerged but a novel polymorphism was identified in the promoter region. Overall, our results suggest that germline mutations of NFKBIA are not a significant cause of familial aggregation of HL but may contribute to inherited susceptibility to HL.

LRF Virus Centre, Institute of Comparative Medicine, University of Glasgow, Glasgow

Marafioti T, Mancini C, Ascani S, et al.
Leukocyte-specific phosphoprotein-1 and PU.1: two useful markers for distinguishing T-cell-rich B-cell lymphoma from lymphocyte-predominant Hodgkin's disease.
Haematologica. 2004; 89(8):957-64 [PubMed]

BACKGROUND AND OBJECTIVES: T-cell-rich B-cell lymphoma is a rare variant of diffuse large B-cell lymphoma. It shows morphologic, phenotypic and molecular similarities to lymphocyte predominant Hodgkin's disease, and in consequence the two diseases may sometimes be difficult to distinguish. In this paper, we have evaluated the usefulness of the pan-leukocyte marker LSP1 and the transcription factor PU.1 for resolving such diagnostic problems.
DESIGN AND METHODS: Immunohistochemical techniques were used to investigate the expression of LSP1 and PU.1 in 34 tumors, comprising typical examples of T-cell-rich B-cell lymphoma (15 cases), lymphocyte-predominant Hodgkin's disease (13 cases), and lymphocyte-rich classical Hodgkin's disease (6 cases).
RESULTS: The neoplastic cells of T-cell-rich B-cell lymphoma were LSP1-positive and PU.1-negative, whereas the lymphocytic and/or histiocytic (L&H) cells of lymphocyte-predominant Hodgkin's disease were mostly LSP1-negative, with variable PU.1 expression. The two markers did not discriminate between T-cell-rich B-cell lymphoma and lymphocyte-rich classical Hodgkin's disease, whilst they concurred to the distinction between lymphocyte-predominant and lymphocyte-rich classical Hodgkin's disease by integrating the already available tools.
INTERPRETATION AND CONCLUSIONS: Antibodies to LSP1 and PU.1 may represent useful reagents for the differential diagnosis between T-cell-rich B-cell lymphoma and lymphocyte-predominant Hodgkin's disease.

Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford

Dutton A, O'Neil JD, Milner AE, et al.
Expression of the cellular FLICE-inhibitory protein (c-FLIP) protects Hodgkin's lymphoma cells from autonomous Fas-mediated death.
Proc Natl Acad Sci U S A. 2004; 101(17):6611-6 [PubMed] Free Access to Full Article

Hodgkin's lymphoma (HL) is characterized by the presence of malignant so-called Hodgkin's/Reed-Sternberg (HRS) cells, which display resistance to certain apoptotic stimuli, including a lack of sensitivity to Fas-mediated cell death. However, the mechanisms responsible for their resistance to apoptosis inducers have not been elucidated. Here we confirm that both HL-derived cell lines and the HRS cells of primary HL tissues express Fas ligand (FasL) along with the inhibitory c-FLIP protein. Down-regulation of cellular FLICE (FADD-like IL-1beta-converting enzyme)-inhibitory protein (c-FLIP) through the use of specific small inhibitory RNAs (siRNAs) leads to reduced viability of the L428 and L591 HL-derived cell lines. To determine whether endogenous FasL was responsible for the reduction in cell viability observed after down-regulation of c-FLIP, L428 and L591 cells were treated with c-FLIP-specific siRNAs with and without siRNAs directed to FasL. Treatment of these cells with both c-FLIP- and FasL-specific siRNAs in combination restored cell viability to near control levels. Our results provide a mechanism whereby HRS cells are protected from autonomous FasL-mediated cell death while preserving their ability to evade immunosurveillance. Targeting c-FLIP could provide a novel approach to the treatment of HL.

Department of Pathology, University of Birmingham, Birmingham B15 2TT

Marshall NA, Christie LE, Munro LR, et al.
Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma.
Blood. 2004; 103(5):1755-62 [PubMed]

Although immunosuppression has long been recognized in Hodgkin lymphoma (HL), the underlying basis for the lack of an effective immune response against the tumor remains unclear. The aim was to test our hypothesis that regulatory T cells dominate involved lymph nodes. The approach was to assay CD4+ T-cell function in HL-infiltrating lymphocytes (HLILs) and paired peripheral blood mononuclear cells (PBMCs) of 24 patients. Strikingly, unlike PBMCs, HLILs were anergic to stimulation with mitogen, primary, or recall antigens, mounting no proliferative responses and only rare T-helper 1 (Th1) or Th2 cytokine responses. Mixing paired HLILs and PBMCs showed the anergic effect was dominant and suppressed PBMC responses. Furthermore, flow cytometry demonstrated that HLILs contained large populations of both interleukin-10 (IL-10)-secreting T-regulatory 1 (Tr1) and CD4+CD25+ regulatory T cells. We found evidence for 3 mechanisms of action implicated in the suppressive functions of regulatory T cells: the inhibition of PBMCs by HLILs was ameliorated by neutralizing IL-10, by preventing cell-to-cell contact, and by blocking anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4). Thus, HLILs are highly enriched for regulatory T cells, which induce a profoundly immunosuppressive environment and so provide an explanation for the ineffective immune clearance of Hodgkin-Reed Sternberg cells.

Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen

Chui DT, Hammond D, Baird M, et al.
Classical Hodgkin lymphoma is associated with frequent gains of 17q.
Genes Chromosomes Cancer. 2003; 38(2):126-36 [PubMed]

The etiology of Hodgkin lymphoma (HL) is poorly understood, and studies of the genetics of this disease have been hampered by the scarcity of the Hodgkin and Reed-Sternberg (HRS) cells within tumors. To determine whether recurrent genomic imbalances are a feature of HL, CD30-positive HRS cells were laser-microdissected from 20 classical Hodgkin lymphomas (cHLs) and four HL-derived cells lines and subjected to analyses by comparative genomic hybridization. In primary tumors, the most frequently involved chromosomal gains were 17q (70%), 2p (40%), 12q (40%), 17p (40%), 22q (35%), 9p (30%), 14q (30%), and 16p (30%), with minimal overlapping regions at 17q21, 2p23-13, 12q24, 17p13, 22q13, 9p24-23, 14q32, 16p13.3, and 16p11.2. The most frequent losses involved 13q (35%), 6q (30%), 11q (25%), and 4q (25%), with corresponding minimal overlapping regions at 13q21, 6q22, 11q22, and 4q32. Statistical analysis revealed significantly more gains of 2p and 14q in the older adult cases; loss of 13q was associated with a poor outcome. The results suggest that there is a set of recurrent chromosomal abnormalities associated with cHL and provide further evidence that cHL is genetically distinct from nodular lymphocyte predominance Hodgkin lymphoma (NLPHL). Abnormalities of 17q are infrequent in other lymphomas or NLPHL; this finding, coupled with current knowledge of gene expression in cHL, suggests that genes present on 17q may play an important role in the pathogenesis of cHL.

Leukaemia Research Fund Virus Centre, Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Glasgow

Marafioti T, Pozzobon M, Hansmann ML, et al.
Expression of intracellular signaling molecules in classical and lymphocyte predominance Hodgkin disease.
Blood. 2004; 103(1):188-93 [PubMed]

The neoplastic cells in classical Hodgkin disease (Reed-Sternberg cells) are of B-lymphoid origin, but they lack many markers of this cell lineage, for example, immunoglobulin, CD20, and B-cell-associated transcription factors. In contrast, the neoplastic cells ("L&H" cells) in lymphocyte predominance Hodgkin disease retain the molecular profile of germinal center B cells. In this paper, we investigated the expression in Hodgkin disease (45 cases and 3 cell lines) of 5 intracellular signaling molecules found in B cells. The Src family kinase Syk, the B-cell adaptor protein BLNK, and phospholipase C (PLC)-gamma2 were consistently absent from Reed-Sternberg cells, whereas 2 other Src kinases (Lyn and Fyn) were heterogeneously expressed in a proportion of cases (12% and 42%, respectively). In contrast, the tumor cells in all cases of lymphocyte predominance Hodgkin disease were positive for Fyn, Syk, BLNK, and PLC-gamma2, and Lyn immunostaining was seen in a minority of biopsies. These results indicate that in Reed-Sternberg cells, the defect in B-cell lineage marker expression includes a spectrum of molecules involved in intracellular signaling, a finding in keeping with recent gene expression profiling studies. Furthermore, the clear difference in expression of signaling proteins between the 2 major subtypes of Hodgkin disease may be of diagnostic value.

Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford

Olson PD, Yoder K, Fajardo L-G LF, et al.
Lethal invasive cestodiasis in immunosuppressed patients.
J Infect Dis. 2003; 187(12):1962-6 [PubMed]

Using both traditional methods and broad-range 18S ribosomal DNA (rDNA) polymerase chain reaction, we examined 2 cases of lethal cestodiasis, in which the disease agent had been poorly identified or misidentified. In one case, involving a patient with AIDS, we identified the human dwarf tapeworm, Hymenolepis nana, as a cause of aberrant metastatic larval disease. In the second case with similar pathologic abnormalities, involving a patient with Hodgkin disease, we identified a larval cestode with a previously uncharacterized 18S rDNA sequence. A prior report of this case nearly 30 years ago, based on tissue examination, had suggested that the parasite was a sparganum.

Parasitic Worms Division, Department of Zoology, Natural History Museum, London