cHL Cases Research Articles

Epstein-Barr virus (EBV) positive classical Hodgkin lymphoma of Iraqi children: An immunophenotypic and molecular characterization of Hodgkin/Reed-Sternberg cells

Classical Hodgkin lymphoma (cHL) in children is often associated with EBV infection, more commonly in developing countries. Here we describe the histological, immunohistochemical, and molecular features of 57 cases of HL affecting Iraqi children under 14 years of age. Histologically, 51 cases were classified as cHL of Mixed Cellularity and Nodular Sclerosis subtypes (MC = 69%; NS = 31%), and 6 cases as Nodular Lymphocyte Predominant HL (NLP-HL). EBV infection of H/RS cells was demonstrated in 44 of 51 cases of cHL (86%), and was more common in MC than in NS (97% vs. 63%; P = 0.0025). The immunophenotypic profile of H/RS cells was similar in MC and NS, and was not influenced by EBV infection; H/RS cells were consistently positive for PAX-5 and to a lesser degree for other B cell markers including CD20/CD79a, OCT-2, and BOB-1. Clonal IGH rearrangements were detected in 14 of 38 cHL (37%), with no significant difference between MC and NS cases, and with no association with the EBV status. Oligoclonal/monoclonal TCRγ rearrangements were present in 28 of 38 cases (74%), suggestive of restricted T cell responses. Our findings indicate that cHL occurring in Iraqi children is characterized by immunohistochemical and molecular features undistinguishable from those present in cHL occurring elsewhere in the world. Moreover, the high incidence of EBV-infected H/RS cells and frequent occurrence of restricted T cell responses might be indicative of a defective local immune response perhaps related to the very young age of the children.

PML Nuclear Bodies and SATB1 Are Associated with HLA Class I Expression in EBV+ Hodgkin Lymphoma

Tumor cells of classical Hodgkin lymphoma (cHL) are characterized by a general loss of B cell phenotype, whereas antigen presenting properties are commonly retained. HLA class I is expressed in most EBV+ cHL cases, with an even enhanced expression in a proportion of the cases. Promyelocytic leukemia protein (PML) and special AT-rich region binding protein 1 (SATB1) are two global chromatin organizing proteins that have been shown to regulate HLA class I expression in Jurkat cells. We analyzed HLA class I, number of PML nuclear bodies (NBs) and SATB1 expression in tumor cells of 54 EBV+ cHL cases and used 27 EBV− cHL cases as controls. There was a significant difference in presence of HLA class I staining between EBV+ and EBV− cases (p<0.0001). We observed normal HLA class I expression in 35% of the EBV+ and in 19% of the EBV− cases. A stronger than normal HLA class I expression was observed in approximately 40% of EBV+ cHL and not in EBV− cHL cases. 36 EBV+ cHL cases contained less than 10 PML-NBs per tumor cell, whereas 16 cases contained more than 10 PML-NBs. The number of PML-NBs was positively correlated to the level of HLA class I expression (p<0.01). The percentage of SATB1 positive cells varied between 0% to 100% in tumor cells and was inversely correlated with the level of HLA class I expression, but only between normal and strong expression (p<0.05). Multivariable analysis indicated that the number of PML-NBs and the percentage of SATB1+ tumor cells are independent factors affecting HLA class I expression in EBV+ cHL. In conclusion, both PML and SATB1 are correlated to HLA class I expression levels in EBV+ cHL.

Loss of utility of bone marrow biopsy as a staging evaluation for Hodgkin lymphoma in the positron emission tomography–computed tomography era: a West of Scotland study

Positron emission tomography–computed tomography (PET-CT) scanning has been shown to be more sensitive than bone marrow biopsy (BMB) in detecting bone marrow involvement (BMI) in classical Hodgkin lymphoma (cHL). In this 5-year retrospective series, 93 (54%) of all new cases of cHL were staged using PET-CT and BMB. PET-CT identified focal bone marrow uptake in 17/93 (18.3%), whilst only five of these (29.4%) were confirmed by BMB. Abnormal pelvic uptake was seen on PET-CT in these five cases. The other 12 cases were missed, giving BMB a sensitivity of 29.4% and a specificity of 100%. PET-CT upstaged 9.7% of patients compared to CT alone. BMB upstaged only one patient; however, this patient was already stage IV on PET-CT. BMB did not alter clinical management in any case. Bone positivity on PET-CT appeared to correlate with anemia, raised lactate dehydrogenase (LDH) and B symptoms. BMB has little or nothing to offer staging cHL in the PET-CT era.

P3.248 The Evolution of Sexually Transmitted Infection in Romania

Objectiveto analyse the evolution of incidence of some sexually transmitted infections (STI) in Romania in the transition period.MethodsThe study is based on data recorded at Dermato-venereological Center Bucharest. We had...

I. Pathological and clinical diversity in diffuse large B‐cell lymphoma

I. Pathological and clinical diversity in diffuse large B‐cell lymphoma

Abstract 820: Genome-wide aberration analysis of rare cancer cell populations derived from formalin fixed paraffin embedded tissue.

Abstract Introduction. The majority of clinically described patient material worldwide is available as formalin fixed parafin embedded (FFPE) tissue blocks. Despite being a cheap and convenient way of archiving tissue suitable for review under the microscope, FFPE blocks are difficult to interrogate with modern molecular biology methods due to changes occurring during the process of fixation, embedding and aging. Genetic analysis of tumours with limited malignant cell count, like classical Hodgkin lymphoma (cHL) or T-cell/histiocyte-rich B cell lymphoma (TCHRBCL) is impeded by a bulk mass of non-malignant tumour infiltrating cells. Laser capture microdissection is currently a method of choise for rare tumor cell isolation, however it induce serious damage for gDNA, therefore additional techniques for enrichment are needed to enable meaningful genome-wide aberration analysis of tumors with low cancer cell content. Methods. For extraction of nuclei from FFPE blocks of cHL and TCHRBCL cases enzymatic digestion and mechanical force were utilized. Pure populations of cancer cell nuclei were isolated by fluorescence-activated cell sorting (FACS) based on cancer type-specific nuclear markers Pax-5, Mum-1, Bcl-6, Ki-67. Array comparative genomic hybridization (aCGH) was utilized for genome-wide aberration analysis. Results. We have optimized a set of methods to successfully isolate intact, high quality nuclei from FFPE tissue blocks. Nuclei retain their antigenicity and, therefore, are suitable for labelling with frourophore-conjugarted antibodies, which enables flow-sorting based not only on physical parameters and DNA content, but also on positivity for highly specific nuclear markers. Tumour nuclei populations from cHL and TCHRBCL of 70-90% in purity were isolated. DNA extracted from as little as 500 flow-sorted nuclei was sufficient to generate high-resolution and reproducible aCGH data. Conclusions. Our proposed methodology allows the use of tissues in FFPE blocks more widely. Efficient isolation of malignant cells from the tumour mass was achieved. In the cases where starting material is scarce and very low number of target nuclei can be isolated, DNA extraction with improved recovery together with commercially available whole genome amplification methods enable genome-wide aberration analysis. Citation Format: Darius Juskevicius, Christian Ruiz, Tanja Dietsche, Alex Rufle, Michael T. Barrett, Lukas Bubendorf, Stephan Dirnhofer, Alexander Tzankov. Genome-wide aberration analysis of rare cancer cell populations derived from formalin fixed paraffin embedded tissue. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 820. doi:10.1158/1538-7445.AM2013-820

Composite diffuse large B-cell lymphoma and classical Hodgkin’s lymphoma of the stomach: Case report and literature review

The combination of classical Hodgkin's lymphoma (cHL) and non-Hodgkin lymphoma coexisting in the same patient is not common, especially in one extranodal location. Here we present a rare case of composite diffuse large B-cell lymphoma (DLBCL) and cHL occurring simultaneously in the stomach of a 53-year-old female who presented with upper abdominal discomfort and gas pain. Surgery was performed and the disease was diagnosed pathologically as composite lymphoma of DLBCL and cHL using hematoxylin-eosin and immunohistochemical staining. Epstein-Barr virus (EBV) infection was not detected by in situ hybridization for EBV-encoded RNA or immunohistochemistry for EBV latent membrane protein-1. Polymerase chain reaction analysis from the two distinct components of the tumor demonstrated clonal immunoglobulin κ light chain gene rearrangements. The patient died approximately 11 mo after diagnosis in spite of receiving eight courses of the CHOP and two courses of the rituximab-CHOP (RCHOP) chemotherapy regimen. This case report showed that the two distinct components, DLBCL and cHL, appeared to originate from the same clonal progenitor cell, and that EBV infection was not essential for transformation during the course of tumorigenesis.

Strong HLA Class I Expression Is Positively Correlated with the Number of PML Nuclear Bodies and Negatively with the Percentage of SATB1 Positive HRS Cells in EBV+ Classical Hodgkin Lymphoma (cHL)

AbstractAbstract 3633 Introduction:Classical Hodgkin lymphoma (cHL) is a malignant neoplasm of the immune system, characterized by the presence of an abundant reactive infiltrate and a minority of Hodgkin Reed-Sternberg cells (HRS cells). HRS cells retain their professional antigen presenting phenotype in most cases. In EBV- cHL, membranous HLA class I expression is retained in HRS cells in approximately 30% of the cases, whereas in EBV+ cHL HLA class I expression is retained in HRS cells in 79% of the cases. Moreover, a proportion of the EBV+ cHL cases shows an enhanced HLA class I expression as compared to the surrounding infiltrating cells.The mechanism of the enhanced or lost HLA class I expression is unknown. Special AT-rich region binding protein 1 (SATB1) and promyelocytic leukemia protein (PML) are two proteins that have been shown to regulate HLA class I expression. Downregulation of SATB1 in Jurkat cells results in an enhanced expression of HLA-A, HLA-G, HLA-H and HCG4P6, whereas downregulation of PML results in a reduced HLA-A and HLA-G expression. PML is the main component of nuclear bodies (NBs) that organize the chromatin structure into loops by anchoring matrix attachment regions to the nuclear matrix. SATB1 has been shown to be associated with the PML-NBs in the HLA region. Aim:To investigate the possible role of SATB1 and PML-NBs in the regulation of HLA class I expression in EBV+ cHL. Methods:We analyzed 64 EBV+ cHL cases and as a control included 29 EBV- cHL cases. HLA class I membranous staining (HC10 antibody) by HRS cells was scored as positive or strongly positive, cases that lacked membrane staining were scored negative. ß2-microglobulin served as an additional marker for membranous HLA class I expression. For SATB1 (14/SATB1), we scored the percentage of HRS cells with nuclear SATB1 staining. For PML (PG-M3), we scored HRS cells based on the number of PML nuclear bodies in two categories; 10 or less NBs per cell or >10 NBs per cell (per 4 um tissue section). Results:In the EBV+ group 24 cases stained strongly positive, 22 positive and 18 negative for HLA class I. In the EBV- group, none of the cases showed a strong positive staining, 7 cases stained positive and 22 cases were negative for HLA class I. HLA class I staining results were consistent with the ß2-microglobulin staining results in all cases. The percentage of SATB1 positive HRS cells varied from 0–100% in both EBV+ and EBV- cHL. The number of PML-NBs was between 0–10 in 46 and >10 in 18 EBV+ cHL cases and between 0–10 in 23 and >10 in 6 EBV- cHL cases.We observed no correlation between HLA class I staining in the EBV- cHL cases and the percentage of SATB1 positive HRS cells or the number of PML-NBs in the HRS cells. In EBV+ cHL cases we observed significant differences in the percentages of SATB1 positive cells between the HLA class I negative, normal and strongly positive groups (p=0.0412). The cases with normal HLA class I staining had significantly higher percentages of SATB1 positive cells as compared to the cases with strong HLA class I staining pattern (p<0.05) (Figure 1). There was no significant difference between negative and normal or negative and strongly positive HLA class I groups with respect to the percentage of SATB1. The percentage of EBV+ cHL cases with >10 PML-NBs significantly increased from HLA class I negative (1 out of 18, i.e. 5%) to normal (4 out of 22, i.e. 18%) and strong (13 out of 24, i.e. 54%) positive cHL cases (p=0.0011). Conclusion:We found an inverse correlation between the percentages of SATB1 positive HRS cells in the HLA class I strong and normal EBV+ cHL groups. The number of cases with >10 PML-NBs were significantly increased in HLA class I strong as compared to the HLA class I normal and negative EBV+ cHL groups. Thus SATB1 and PML may play an important role in the regulation of HLA class I expression levels in EBV+ cHL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

HLA-A*02:01 Is Associated with Inferior Overall Survival in EBV-Associated Classical Hodgkin Lymphoma

AbstractAbstract 1526 Introduction:The risk of developing Epstein-Barr Virus (EBV)-associated classical Hodgkin Lymphoma (cHL) shows an association with class I human leukocyte antigen (HLA) genes. HLA-A*01:01 is associated with increased risk of EBV-associated cHL whereas HLA-A*02:01 is associated with decreased risk. (Hjalgrim et. al, PNAS, 2010).The effects of these two alleles are independent of each other and the effect associated with each allele is additive i.e., possession of two HLA-A*01:01 alleles is associated with greater risk than one allele. HLA-A genes are involved in cytotoxic T cell (CTL) responses to exogenous antigens, and these data suggest that EBV-specific CTL responses are important in the pathogenesis of EBV-associated cHL. This study aimed to determine if HLA-A*01:01 and A*02:01 alleles are a factor in determining clinical outcome in EBV-associated cHL. Methods:Patients with cHL were selected from two previous studies: the Scotland and Newcastle Epidemiological Study of Hodgkin’s disease (SNEHD) (Jarrett et. al, Blood, 2005) and the “Investigation of the pathogenesis of Hodgkin lymphoma” (IPHL), an ongoing collection of patients principally from the West of Scotland. Both studies have ethical approval. Clinical follow-up data were obtained through clinical records, liaison with treating physicians or the Scotland and Newcastle Lymphoma Group (SNLG) database. Clinical treatment decisions were made by individual physicians. Cases were selected on the basis of availability of tumour EBV status, a suitable sample for HLA typing and clinical follow-up data and included 257 cases from the SNEHD study and 128 from the IPHL. HLA genotyping was performed to an intermediate resolution using allele-specific PCR (Gen-probe) and subsequent bead-based sequence specific oligonucleotide (SSO) assay (Luminex). Allele assignment was performed using Quicktype for Lifematch software (Gen-probe). Results were analysed for all cases, EBV-associated cases only, and EBV-negative cases only. Overall survival (OS) and Event-free survival (EFS) were analysed by number of HLA-A*01:01 alleles, number of HLA-A*02:01 alleles, and by the presence or absence of HLA-A*01:01 or HLA-A*02:01. Statistical analyses used Kaplan-Meier survival and Cox-regression analyses, adjusting for histological subtype, clinical stage, age and sex, and were implemented using SPSS v.19. Results:385 cases diagnosed between 1993 and 2008 were included in the analysis. 55.8% of cases were male and 128 (33.2%) had EBV-associated disease. Median follow up was 74 months (range 0–220 months). Clinical stage was available for 289 patients: stage 1 (17.9%); stage 2 (41.8%); stage 3 (22.5%); and stage 4 (17.6%). In the analysis of all cases there were no significant differences in OS and EFS by number or presence of HLA-A*02:01 and A*01:01 alleles. EBV-associated cHL cases demonstrated inferior OS with the presence of HLA-A*02:01 (p=0.04) (figure). This was corroborated by the demonstration of inferior OS with increasing number of HLA -A*02:01 alleles present (p=0.016). Increasing number of HLA -A*02:01 alleles was also associated with inferior EFS but differences were not statistically significant. HLA-A*01:01 was not associated with OS or EFS in EBV-associated cHL. There was no significant association between histological subtype and OS or EFS. When clinical stage, age, and sex were included in the Cox-regression analysis of survival in EBV-associated cases, only age (p<0.001) was significant; presence of an HLA-A*02:01 allele approached statistical significance (p =0.062). Conclusions:In this study the HLA-A*02:01 allele was associated with inferior survival in EBV-associated cHL. As HLA-A*02:01 is associated with a decreased risk of developing EBV-associated cHL, we hypothesise that cHL arising in this 'protected' group is associated with greater biological or immunological dysfunction, resulting in poorer outcome. HLA-A genotype may be helpful in identifying patients who have inferior outcomes using standard therapy, and who may benefit from novel or cellular therapies. Larger studies within the context of clinical trials are required to extend these findings. In addition, this study supports the importance of stratifying cHL cases by EBV-status in clinical studies. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Prognostic Value of Six Germline Single Nucleotide Polymorphisms At the REL, HLA-DRA, GATA3 and PVT1 Loci Identified in a Classical Hodgkin Lymphoma Genome-Wide Association Study: A Meta-Analysis of 601 Patients for Progression-Free Survival From Two Independent Studies

AbstractAbstract 3637 Background:A recent large genome-wide association study confirmed the important role of the major histocompatibility complex in classical Hodgkin Lymphoma (cHL) susceptibility (rs6903608), but also identified significant associations with genetic variants outside of the HLA region at chromosomal regions 2p16.1 (REL, rs1432295), 8q24.21 (PVT1, rs2608053, rs2019960), and 10p14 (GATA3, rs501764, rs485411). These single nucleotide polymorphisms (SNPs) are localized in or near important genes involved in lymphoma pathogenesis: REL, which encodes for c-rel, is a member of NFkB pathway; PVT1, which is downstream of the MYC locus, encodes for non-coding microRNAs, some of which have oncogenic properties; and GATA3, which is a critical transcriptional factor for priming lymphoid T-cells for Th2 phenotype, is aberrantly expressed in HL. We investigated whether these six SNPs described in cHL etiology also influence the outcome of newly diagnosed cHL patients in two independent cohorts. Patients and Methods:The first cohort consisted of 342 cHL patients from the Groupe d’Etude des Lymphomes de l’Adulte (GELA) who were prospectively enrolled between 1998 and 2002. The second cohort consisted of 259 patients prospectively enrolled in the University of Iowa/Mayo Clinic SPORE Molecular Epidemiology Resource (MER). DNA for genotyping was extracted from a blood samples collected at diagnosis, and genotyping was conducted using Taqman technology (ABI Prism 7000, Applied Biosystems) for HLA-DRA, PVT1, GATA3 and PCR-based restriction fragment length polymorphism for REL in GELA cohort and Illumina iSelect for the 6 SNPs in MER cohort. Both cohorts were followed for disease progression, re-treatment and death. In the GELA series, the median age was 32 years (range, 15–93), 57% were male, and 72% had an Ann Arbor stage I–II. Histology was nodular sclerosis in 84%, and 29% of available cases (n=40/140) were EBV positive. In the MER series, the median age was 38 years (range, 18–89), 53% were male, and 53% had an Ann Arbor stage I-II. Nodular sclerosis was the most common subtype (62%) and 23 of 115 available cases were EBV positive (20%). The International Prognostic Score (IPS) was 0–1 in 46% and 52% of GELA and MER patients, respectively. ABVD regimen was used for 60% and 88% of the GELA and MER patients, respectively. The 5-year progression free survival (PFS) was 83% and 75% in GELA and MER cohorts, respectively. We estimated the prognostic value of each individual SNP for PFS for allelic and genotypic (dominant and recessive) modes of transmission in each series. In order to increase power, we then performed a meta-analysis of the two studies. Results:Consistent with prior reports, the minor allele frequencies for REL (rs1432295), HLA-DRA (rs6903608), PVT1 (rs2608053), PVT1 (rs2019960), GATA3 (rs501764) and GATA3 (rs485411) were 0.44, 0.50, 0.43, 0.29, 0.22, 0.28 in the GELA and 0.46, 0.41, 0.46, 0.27, 0.25, 0.31 in the MER series, respectively. In GELA, GATA3 (rs501767) was associated with PFS in the ordinal model (HR=1.66, 95%CI 1.11–2.49, P=.01), while there were suggestive associations for PVT1 (rs2608053) (HR=1.76, 95%CI 0.95–3.26, P=.07) and REL (rs1432295) (HR=0.63, 95%CI 0.37–1.06, P=.08) in the dominant model. In the MER, PVT1 (rs2608053) was associated with PFS in the ordinal model (HR=1.42, 95%CI 0.99–2.03, P=.06), while there was a suggestive association with HLA-DRA (rs6903608) in the dominant model (HR=0.64, 95%CI 0.39–1.05, P=.08) for whole cohort but also for EBV negative cHL (HR=0.41, 95%CI 0.17–0.99, P=.05). In a meta-analysis of the two studies, PVT1 (rs2608053) was significantly associated with PFS in the ordinal (HR=1.34, 95%CI 1.04–1.73, P=.02) and the dominant (HR=1.88, 95%CI 1.20–2.95, P=.006) models. Further adjustment for IPS had minimal impact on these associations (ordinal HR=1.30, 95%CI 1.00–1.70; dominant HR=1.78, 95%CI 1.13–2.83). Patients with PVT1(rs2608053) GG, GA, AA genotypes had a 5-year PFS rate of 83%, 74% and 73% in GELA and 86%, 70%, and 73% in the MER, respectively. Conclusions:The non-HLA cHL susceptibility locus PVT1 (rs2608053) was associated with PFS in two independent cohorts of cHL cases, and after adjustment for the IPS. While associations with GATA3 and REL were only observed in one of the studies, their evaluation in other studies is warranted. Functional studies of PVT1 in cHL pathogenesis should be pursued. Disclosures:No relevant conflicts of interest to declare.

Epstein-Barr Virus Status Correlates with Composition and Prognostic Impact of the Tumor Microenvironment in Classical Hodgkin Lymphoma

AbstractAbstract 52 Background:Classical Hodgkin lymphoma (cHL) is often characterized by a minority of neoplastic cells surrounded by a heterogeneous background of reactive non-neoplastic cells. An increased amount of certain cell subsets, such as T-regulatory lymphocytes and macrophages, in the microenvironment of cHL tumor lesions has been found to correlate with an adverse prognosis, probably as a result of enhanced immune tolerance towards tumor cells. Furthermore, it has also been suggested that the presence of Epstein-Barr virus (EBV) infection in the Hodgkin Reed-Sternberg (HRS) cells, may modulate the composition of the tumor microenvironment. Aim:In the present study, we have analyzed the possible correlation between EBV-status, a number of tumor microenvironment parameters and outcome in a large retrospective series of newly diagnosed cHL patients. Design and Methods:A tissue microarray was constructed from paraffin embedded pre-therapeutic tumor tissue biopsies obtained from 288 newly diagnosed cHL cases. The expression in the tumor microenvironment of the macrophage markers CD68 and CD163, the regulatory T-cell marker FoxP3 and the cytotoxic T-cell marker Granzyme-B (GrB) was assessed by immunohistochemistry (IHC) using a previously described semi-automated stereological counting approach (Haematologica 2011;96:269–276). The presence of EBV in HRS cells was investigated using ’in situ’ hybridization for EBV-encoded RNAs 1 and 2 and LMP-1 IHC. Clinical data were obtained from clinical records. The correlation between clinic-pathological features and EBV was assessed using the rank-sum or Kruskal-Wallis test. The impact of clinico-pathological parameters on event-free (EFS) and overall survival (OS) was evaluated using the log rank test. Results:The 288 patients had a median age of 37 yrs (range: 6–86 yrs). The M:F ratio was 1.3. One third (33%) of the patients were positive for EBV in the HRS cells. EBV-positive cases exhibited higher numbers of CD68 (p=0.001), CD163 (p=0.0002), GrB (p<0.0001), and FoxP3 (0.0009)-positive cells. Excluding cases of mixed cellularity from the analysis, the significant correlation between EBV and CD163 (p=0.03), GrB (p=0.003), FoxP3 (p=0.006) remained, whereas the correlation for CD68 was slightly weakened (p=0.06).In the entire cohort (n=288), a high expression of CD68, CD 163 and GrB were found to correlate with significantly lower OS and EFS (high vs. low CD68: 5-year OS, 73% vs. 87% p=0.002, 5-year EFS, 58% vs. 70% p=0.03; high vs. low CD163: 5-year OS, 78% vs. 87%, p=0.03, 5-year EFS, 62% vs. 69%, p= 0.04) and high GrB: 5-year OS, 77% vs 88 %, p=0.004, 5-year EFS, 61% vs. 69%, p= 0.02). Interestingly, the influence of tumor microenviromental parameters on outcome was more pronounced in EBV-negative cases (n=193) than in EBV-positive ones (n=95). In the former, significantly lower OS and EFS values were associated with a high expression of CD68 (high vs. low CD68: 5-year OS, 60% vs. 92%, 5-year EFS, 43% vs. 71%, both p<0.001), high CD163 (5-year OS, 72% vs. 89%, p<0.001, 5-year EFS, 58% vs. 69%, p= 0.03) and high GrB (5-year OS, 61% vs. 90%, 5-year EFS, 43%vs. 71%, both p<0.001). Among the EBV-positive cohort, the corresponding OS and EFS values were high CD68 (5-year OS, 85% vs. 82%, p=0.69, 5-year EFS, 72%vs. 66%, p=0.43), high CD163 (5-year OS, 86% vs. 84%, p=0.34, 5-year EFS, 67%vs. 71%, p=0.48) and high GrB (5-year OS, 88% vs. 81%, p=0.34, 5-year EFS, 73%vs. 65%, p= 0.63).The number of FoxP3-cells was not found to affect the prognosis in neither EBV-negative nor EBV-positive cases. Conclusions:The present study confirms that the EBV-status in cHL is associated with distinct features of the tumor microenvironment. As a novel finding, our results suggest that the prognostic impact of intratumoral reactive non-neoplastic cell subsets is EBV-status dependent, i.e. a significantly adverse impact of an increased amount of certain bystander cell subsets on outcome was only found in EBV-negative cases. Disclosures:No relevant conflicts of interest to declare.

Expression, Prognostic Value and Function of IGF1R in Hodgkin Lymphoma.

AbstractAbstract 2640 Background:Classical Hodgkin's lymphoma (cHL) is a B cell lymphoma characterized by a minority of malignant Hodgkin Reed-Sternberg (HRS) cells that are surrounded by a vast majority of reactive cells. These reactive cells provide a microenvironment that supports survival and growth of the HRS cells. The interaction between cancer cells and microenvironment includes aberrant activity of multiple receptor tyrosine kinases (RTK). The insulin-like growth factor-1 receptor (IGF1R) is a RTK that is involved in many malignancies and plays a crucial role in proliferation, transformation, apoptosis protection and chemotherapy-resistant. In this study we evaluated the expression, prognostic significance and function of IGF1R in cHL. Design and Methods:Immunohistochemistry was applied on 80 cHL cases. IGF1R expression was correlated with clinical data in a cHL patient cohort using Cox-regression, Chi-square and one-way ANOVA statistics. Western blot was used to determine IGF1R expression in four cHL cell lines (L428, L1236, KMH2 and UHO-1) and the IGF1 production was measured by ELISA. The Alamar blue assay was performed to determine the effect of IGF1 and cyclolignan picropodophyllin (PPP, a highly selective IGF1R inhibitor) treatment on cell growth. Flowcytometry was performed to analyze cell cycle distribution after PPP treatment. Results:Expression of IGF1R was observed in all or the vast majority of HRS cells in positive cases, whereas in negative cases almost all HRS cells were negative. Expression of IGF1R was detected in 55% (44/80) of the cHL patients. There was a significant difference in IGF1R expression with subtype (p=0.0157), with a high percentage of positive cases in the nodular-sclerosis cases (38/61) and a low percentage of positive cases in mixed-cellularity cases (1/9). There was no association between IGF1R expression and age, EBV status or Ann Arbor Stage. The 5-year progression-free survival (PFS) was 95% in IGF1R positive patients, whereas in IGF1R negative patients the 5-years PFS was 77% (p=0.018).All four cHL cell lines showed expression of IGFR1, with the highest levels observed for L428 cells. The IGF1 production level was similar for all four cell lines. IGF1 treatment had the most prominent effect (31% increase) on the proliferation of L428 cells, whereas only a small effect was seen in the other three cell lines. Thus L428 cells were most sensitive to IGF1 treatment, consistent with its high IGF1R expression level. After 72 hours of PPP treatment (2μM) both the viability and proliferation decreased in all 4 cHL cell lines. The strongest effects were observed for L428 cells and UHO-1 cells with a reduction in cell growth of 48% and 43% respectively. The reduction in cell growth was 30% in KM-H2 cells and 26% in L1236. Treatment with PPP induced a G2/M cell cycle arrest in all 4 cell lines. After 24 hours incubation with PPP (2μM), the G2/M cell population increased from 8% to 45% in L428, from 10% to 64% in UHO-1, from 4% to 48% in KMH2 and from 3% to 38% in L1236. Conclusions:Expression of IGF1R in the HRS cells was observed at a high frequency in the nodular sclerosis subtype and correlates with a favorable 5-years PFS. Functional studies in cHL showed a marked decrease in cell growth and an increase in the percentage of cells in the G2/M phase in all four cHL cell lines upon inhibition of IGF1R. Disclosures:No relevant conflicts of interest to declare.

Tumor-Associated Macrophages in Pediatric Classical Hodgkin Lymphoma: Association with Epstein-Barr Virus, Lymphocyte Subsets, and Prognostic Impact

Tumor-infiltrating macrophages are associated with adverse outcome in adult classical Hodgkin lymphoma (cHL). We have previously shown age-related changes in the lymphocyte composition of pediatric cHL. We therefore hypothesized that the number, function, and prognostic impact of macrophages in pediatric cHL would be different from adult cases. We analyzed the number of macrophages and dendritic cells (DC) in the tumor microenvironment of pediatric cHL by immunohistochemistry. Results were analyzed in context of age, histologic characteristics, Epstein-Barr virus (EBV) status, clinical follow-up, and our previous study of T-cell populations in these cases. One hundred cHL cases were studied, including 69% nodular sclerosis and 23% mixed cellularity cases. A total of 44.8% of cases were EBV-positive. Patients ≤10 years displayed more CD14(+) cells (P = 0.025). In comparison with nodular sclerosis, mixed cellularity was characterized by higher numbers of CD14(+), (P = 0.003) and CD163(+) cells (P = 0.027). EBV(+) cases exhibited higher numbers of CD14(+) (P < 0.0005), CD68(+) (P = 0.005), and CD163(+) cells (P = 0.02). CD68-positive cells did not display an effect on outcome. Worse overall survival was observed in cases with CD163/CD8 ratio ≥2 (P = 0.007). High numbers of CD163(+) cells were associated with worse progression-free survival (PFS; P = 0.015). Furthermore, high numbers of CD163(+) and granzyme B(+) cells were associated with worse PFS in EBV-negative (P = 0.005) but not in EBV-positive cases. Our results suggest that macrophage composition in pediatric cHL is distinct from adults. Functional status of macrophages and their value as prognostic indicators in pediatric cHL may depend on EBV status.

FOXO1 is a tumor suppressor in classical Hodgkin lymphoma

AbstractThe FOXO transcription factors control proliferation and apoptosis in different cell types. Their activity is regulated by posttranslational modifications, mainly by the PI3K-PKB pathway, which controls nuclear export and degradation. We show that FOXO1 is highly expressed in normal germinal center B cells as well as in non-Hodgkin lymphomas, including follicular lymphoma, diffuse large B-cell lymphoma, mucosa-associated lymphoid tissue non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, and mantle cell lymphoma. In contrast, in 31 of 32 classical Hodgkin lymphoma (cHL) cases, Hodgkin and Reed-Sternberg cells were FOXO1 negative. Neoplastic cells of nodular lymphocyte-predominant Hodgkin lymphoma were negative in 14 of 20 cases. FOXO1 was down-regulated in cHL cell lines, whereas it was expressed in non-Hodgkin lymphoma cell lines at levels comparable with normal B cells. Ectopic expression of a constitutively active FOXO1 induced apoptosis in cHL cell lines and blocked proliferation, accompanied with cell-cycle arrest in the G0/G1 phase. We found that, in cHL cell lines, FOXO1 is inactivated by multiple mechanisms, including constitutive activation of AKT/PKB and MAPK/ERK kinases and up-regulation of microRNAs miR-96, miR-182, and miR-183. These results suggest that FOXO1 repression contributes to cHL lymphomagenesis.

Genetic lesions of the TRAF3 and MAP3K14 genes in classical Hodgkin lymphoma

Hodgkin and Reed/Sternberg (HRS) cells in classical Hodgkin lymphoma (cHL) show constitutive activation of nuclear factor (NF)-κB. Several genetic lesions contribute to this deregulated NF-κB activity. Here, we analysed two further NF-κB regulators for genetic lesions, the inhibitory factor TRAF3 and the key signalling component of the alternative NF-κB pathway, MAP3K14 (NIK). Single nucleotide polymorphism (SNP) array analysis of cHL cell lines revealed a uniparental disomy of the long arm of chromosome 14 associated with a biallelic deletion of TRAF3 located on this chromosome in cell line U-HO1. Cloning of the deletion breakpoint showed a 123371 bp deletion. No inactivating mutations of TRAF3 were found in six other cHL cell lines or in microdissected HRS cells from seven cHL. However, in primary cHL samples interphase cytogenetic analyses revealed signal patterns indicating monoallelic deletion of TRAF3 in 3/20 other cases. SNP array analysis revealed a gain of copy number for MAP3K14 in three cHL cell lines. Gains of MAP3K14 were detected in 5/16 cases of primary cHL. In conclusion, in rare instances, HRS cells harbour inactivating mutations of the TRAF3 gene and recurrently show gains of MAP3K14, indicating that more components of NF-κB signalling show genetic lesions in HRS cells than previously known.

HLA-A*02:07 Is a Protective Allele for EBV Negative and a Susceptibility Allele for EBV Positive Classical Hodgkin Lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10−5). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population.

Improved clonality detection in Hodgkin lymphoma using the BIOMED‐2‐based heavy and kappa chain assay: a paraffin‐embedded tissue study

Although BIOMED-2 polymerase chain reaction (PCR) standardization protocols allow clonality detection in nearly 100% of non-Hodgkin B cell lymphomas, they have not been widely validated for Hodgkin lymphoma (HL). Our aim was to assess BIOMED-2 protocol sensitivity when using non-microdissected, formalin-fixed, paraffin-embedded (FFPE) tissue from HL cases. We studied 69 consecutive HL cases, of which 61 corresponded to classic HL (cHL) and eight to nodular lymphocyte-predominant HL (NLPHL). CD30-positive cell numbers (<10, 10-25 or >25 per ×200 field), background CD20-positive cell density (low or high) and tumour cell immunophenotype were evaluated. IGH and IGK clonality was assessed on FFPE tissue following BIOMED-2 protocols. Of the 58 assessable cHL cases, 15 (25.9%) exhibited IGH and/or IGK clonality; IGH clonality was shown by nine (15.5%) and IGK clonality by 12 (20.7%). Clonality detection rates in cHL improved as CD30-positive Reed-Sternberg (RS) cell density increased and CD20-positive B cell density decreased, although these correlations did not reach statistical significance. Of the eight NLPHL cases studied, none showed clonal rearrangement. Combined study of IGH and IGK rearrangement according to BIOMED-2 protocols improves clonality detection rate (up to 25% of cases) in HL, even when working on non-microdissected FFPE tissue.

Multifunctional immune-modulatory protein HLA-E identified in classical Hodgkin lymphoma: Possible implications

Although the role of the non-classical human leukocyte antigen E (HLA-E) was originally thought to be limited to the development of a maternal tolerance to a semiallogeneic fetal graft, it is now known that HLA-E exerts multiple immunoregulatory functions. The significance of the presence of HLA-E in neoplastic cells and/or backround microenvironment cells in classical Hodgkin lymphoma (cHL) is not well characterized. In our study, we evaluated the presence of HLA-E in both neoplastic and background cells in 40 cases of cHL. We found HLA-E in both neoplastic and background cells. There was a positive statistical correlation between the expression of HLA-E in neoplastic cells and the clinical stage of the disease, which indicates an immune-tolerogenic property of this multiple-purpose molecule. The presence of HLA-E in background cells seems to be prognostically neutral but its significance still needs to be determined. The mechanisms regulating the immunopathology of cHL neoplastic cells with respect to the presence of these molecule deserve further studies.

CD99 triggers upregulation of miR‐9‐modulated PRDM1/BLIMP1 in Hodgkin/Reed–Sternberg cells and induces redifferentiation

CD99 is a 32-kDa transmembrane glycoprotein that is encoded by the MIC2 gene. Our study was carried out to examine the role of CD99 in tumor progression of classical Hodgkin lymphoma (cHL). Here, we showed that lowly expressed CD99 protein in cHL cell lines and primary cHL cases correlates with the deficient expression of the positive regulatory domain 1 (PRDM1/BLIMP1). In addition, cHL cell lines showed high levels of miR-9 expression. We determined that the upregulation of CD99 induced expression of transcription factor PRDM1, a master regulator of plasma-cell differentiation, which is also a target for miR-9-mediated downregulation. Indeed, inhibition of miR-9 also triggered upregulation of PRDM1 expression. Furthermore, overexpression of CD99 resulted in changed growth features and reorganization of actin cytoskeleton. As upregulation of CD99 led to a decrease in cHL diagnosis marker CD30 and CD15 and an increase in plasma-cell differentiation marker CD38 and the restoration of B-cell makers PAX5, CD79α and CD19, we suggest that downregulated CD99 leads to the prevention of plasma-cell differentiation in Hodgkin/Reed-Sternberg (H/RS) cells. Furthermore, these data indicate that CD99 may control miR-9 expression, which directly targets PRDM1. Altogether, these results reveal a CD99-miR-9-PRDM1 molecule axis in lymphomagenesis of cHL and suggest that upregulation of CD99 in H/RS cells induces terminal B-cell differentiation, which may provide a novel therapeutic strategies for cHL.

JAK2, As Well As PDL1 and PDL2, are Recurrently Targeted by 9p24 Structural and Numerical Aberrations in Lymphoid Neoplasms of Both B- and T-Cell Origin

Abstract 2460 Introduction:Genetic aberrations of the 9p terminal region are recurrent in hematological malignancies. Many of them involve the JAK2 gene located at 9p24, which is non-randomly targeted by chromosomal translocations, amplifications and mutations in various types of leukemia, lymphoma and myeloproliferative neoplasms. Of interest, recent reports showed that also PDL1 and PDL2, two genes located 350kb centromeric to JAK2, are involved in 9p24 aberrations. These genes, coding for the programmed death ligands, were identified as key targets of 9p24 amplification in primary mediastinal B-cell lymphoma (PMBCL) and classical Hodgkin lymphoma (cHL). Moreover, CIITA-PDL1 and CIITA-PDL2 fusions, leading to overexpression of PDL1 and PDL2, respectively, have been reported in two PMBCL cases. Aims:The objective of this study is to characterize structural and numerical 9p24 aberrations in lymphoid malignancies and to assess the molecular consequences of these abnormalities. Study design:Eighty-four cases of B-cell non-Hodgkin lymphoma (B-NHL) and 4 cases of T-cell NHL with 9p24 aberrations were selected for this study. In addition, we included 130 cases of cHL with unknown 9p24 status. All cases were screened by FISH with a dual color break apart assay for JAK2 containing probes covering PDL1 and PDL2. Cases with rearrangements of these loci were further characterized by FISH with BAC and fosmid probes, and by aCGH. In available cases, qRT-PCR and/or IHC was used to determine the expression level of candidate genes. Results:Structural aberrations of 9p24 were identified in 8 patients presenting with CLL in Richter transformation (1), MALT/MZL (2), FL (1), DLBCL (2) and ALL/LL (2). The 9p24 breakpoints were further mapped to a 200 kb region centromeric to JAK2 and harboring PDL1 and PDL2. These aberrations (translocations, insertion, inversion) involved either immunoglobulin (IG) genes (3 cases), or non-IG sequences at 1p34/PHACTR4, 4p13, 3p25, 6q13/EEF1A1 and 13q11 (5 cases, including one with a three-way translocation also involving IGH). In one case with inv(9)(p24p23), a microdeletion affecting the 3' region of JAK2 and the 5' region of PDL1 was detected by FISH and aCGH. The expression pattern of 5 candidate genes analyzed in 5 available cases showed upregulation of PDL1 and PDL2 in 1 case, overexpression of sole PDL2 in 2 cases and a normal level of analyzed transcripts in 2 cases. In both cases with t(9;14)(p24;q32) translocation of JAK2 to der(14) was demonstrated by FISH. Unfortunately, these cases were not available for qRT-PCR.9p24 amplification hallmarked by homogeneously staining region (hsr) was identified in 10 cases of PMBCL and 4 cases of T-NHL. FISH screening of 130 cases of cHL identified 54 lymphomas with low copy number gains (5–10 copies) and 30 cases with 9p24 amplification, including 6 cases with a predominant amplification of the region covering PDL1 and PDL2. In addition, putative involvement of PDL1/PDL2 in unbalanced translocations associated with loss of JAK2 and telomeric 9p24 sequences was found in 3 cases of cHL.Expression of the JAK2, PDL1 and PDL2 mRNA was analyzed in 7 cases of PMBCL and 2 cases of T-NHL. Among the PMBCL samples, 4 showed overexpression of all 3 transcripts, 2 showed upregulation of PDL1 and PDL2, and one showed overexpression of sole PDL1. JAK2 and PDL1 were found to be highly expressed in one case of T-NHL, while a case of ALK+ ALCL revealed only upregulation of PDL1.IHC analysis of P-JAK2, PDL1 and PDL2 proteins in available cases is ongoing. Conclusions:Our study demonstrates that the 9p24 region is recurrently targeted by structural and numerical aberrations in B- and T-cell leukemia/lymphoma. These abnormalities may represent primary as well as secondary genetic changes acquired during progression of the disease. Structural aberrations predominantly involve PDL1 and/or PDL2 that seem to be deregulated by their juxtaposition with regulatory sequences provided by partner genes. Five postulated PDL1/PDL2 partner genes include IGH/14q32, IGL/22q11, PHACTR4/1p34.3 EEF1A1/6q13 and JAK2/9p24. In the analyzed PMBCL and T-NHL cases, 9p24 amplification constantly targets PDL1 and is randomly associated with an aberrant co-expression of either JAK2 or PDL2. Given the role of PDL1/PDL2 in inhibition of T-cell activation, their impaired expression in B- and T-cell lymphoma may lead to “T-cell exhaustion” and deficient cellular immunity favoring tumor progression. Disclosures:No relevant conflicts of interest to declare.