Pathogenesis Of Classical Hodgkin Lymphoma Research Articles

Pinpointing Functionally Relevant miRNAs in Classical Hodgkin Lymphoma Pathogenesis.

Classical Hodgkin lymphoma (cHL) is a hematological malignancy of B-cell origin. The tumor cells in cHL are referred to as Hodgkin and Reed-Sternberg (HRS) cells. This review provides an overview of the currently known miRNA-target gene interactions. In addition, we pinpointed other potential regulatory roles of microRNAs (miRNAs) by focusing on genes related to processes relevant for cHL pathogenesis, i.e., loss of B-cell phenotypes, immune evasion, and growth support. A cHL-specific miRNA signature was generated based on the available profiling studies. The interactions relevant for cHL were extracted by comprehensively reviewing the existing studies on validated miRNA-target gene interactions. The miRNAs with potential critical roles included miR-155-5p, miR-148a-3p, miR-181a-5p, miR-200, miR-23a-3p, miR-125a/b, miR-130a-3p, miR-138, and miR-143-3p, which target, amongst others, PU.1, ETS1, HLA-I, PD-L1, and NF-κB component genes. Overall, we provide a comprehensive perspective on the relevant miRNA-target gene interactions which can also serve as a foundation for future functional studies into the specific roles of the selected miRNAs in cHL pathogenesis.

Tackling the dysregulated immune-checkpoints in classical Hodgkin lymphoma: bidirectional regulations between the microenvironment and Hodgkin/Reed-Sternberg cells.

Immune evasion is considered one of the modern hallmarks of cancer and is a key element in the pathogenesis of classical Hodgkin Lymphoma (cHL). This haematological cancer achieves effective avoidance of the host's immune system by overexpressing the PD-L1 and PD-L2 proteins on the surface of the neoplastic cells. Subversion of the PD-1/PD-L axis, however, is not the sole contributor to immune evasion in cHL, as the microenvironment nurtured by the Hodgkin/Reed-Sternberg cells is a major player in the creation of a biological niche that sustains their survival and hinders immune recognition. In this review, we will discuss the physiology of the PD-1/PD-L axis and how cHL is able to exploit a plethora of different molecular mechanisms to build an immunosuppressive microenvironment and achieve optimal immune evasion. We will then discuss the success obtained by checkpoint inhibitors (CPI) in treating cHL, both as single agents and as part of combination strategies, analysing the rationale for their combination with traditional chemotherapeutic compounds and the proposed mechanisms of resistance to CPI immunotherapy.

Autophagy and cellular senescence in classical Hodgkin lymphoma

Autophagy and cellular senescence are interrelated cellular stress responses important for cellular homeostasis and they have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL). However, the presence of autophagy and cellular senescence and their relation with clinical and laboratory parameters needs further elucidation. Thus, autophagy (LC3B and p62 immunohistochemical expression) and cellular senescence (p16 immunohistochemical expression and SenTraGor™ staining) were studied in tissue sections from 59 patients with cHL. Autophagy and cellular senescence-associated markers were detected in variable proportions of Hodgkin and Reed-Sternberg (HRS) cells in cHL cases. High nuclear p62 immunohistochemical expression in HRS cells showed significant positive correlation with relapse of the disease (p = 0.037). Heterogeneous autophagy and cellular senescence patterns were revealed in HRS cells suggesting biological heterogeneity of cHL. The detection of high nuclear p62 expression in HRS cells may identify a subset of cHL with more aggressive clinical behavior.

The contribution of ebv to the pathogenesis of classical hodgkin lymphoma

: Hodgkin lymphoma (HL) exists in two major forms, the so-called classical type (cHL) and the nodular lymphocyte predominant type (NLPHL). Since NLPHL is considered to be an EBV-negative entity, this review focusses only on the cHL form. Although cHL is a curable disease in many cases, we still lack an understanding of its pathogenesis that could lead to kinder treatments for patients, and also to more effective therapies for the smaller subset of patients who are destined to die of their disease. One approach might be to therapeutically target the Epstein-Barr virus (EBV), which is present in up to one-half of cases in resource rich nations, and in almost all cases in some resource-poor countries. However, it has been suggested that EBV might be simply a silent passenger in cHL. In this review, we present evidence in support of a crucial role for EBV in virus-positive cHL. In particular, we highlight important epidemiological differences between EBV-positive and EBV-negative cHL that suggest different aetiologies, as well as genetic differences, including a different profile of somatic mutations pointing to a distinct contribution for EBV in substituting for cellular genetic changes that are required for disease development when the virus is absent. We also focus attention on important roles for the individual latent virus genes in the pathogenesis of cHL. Overall, this review suggests that a better understanding of how EBV contributes to the pathogenesis of cHL may eventually lead to improved stratification of patients and to the development of therapies that specifically target EBV or its latent genes.

Downregulation of the Tumor Suppressor SAMHD1 in Classical Hodgkin Lymphoma

Introduction: The SAM domain and HD domain 1 (SAMHD1) protein is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase, which has been initially described to restrict human immunodeficiency virus type 1 (HIV-1) in certain cell types through depletion of intracellular dNTP substrates for HIV-1 reverse transcription. Mutations of SAMHD1 gene have been linked to Aicardi-Goutières syndrome (AGS) and have been identified as putative drivers of chronic lymphocytic leukemia resulting in decreased SAMHD1 mRNA and protein levels. Therefore, a line of evidence suggests that SAMHD1 may play a role in oncogenesis as a tumour suppressor. In addition, SAMHD1 may confer resistance to nucleoside-based chemotherapies by hydrolysing their active triphosphate metabolites, and thus, it may lead to resistance to certain chemotherapeutic agents with cytarabine in acute myeloid leukemia being a recent example (Herold et al, Nat Med 2017; 23(2):256-263). The expression patterns and the potential role of SAMHD1 in the pathogenesis of classical Hodgkin lymphoma (HL) are not yet known.Methods: SAMHD1 protein levels were assessed by Western blot analysis in 20 human lymphoma and leukemia cell lines including 5 classical HL (L1236, L428, HDML2, HDMyZ, MDA-V) and 3 diffuse large B-cell lymphoma (DLBCL) (RC-K8, OCI-Ly3, U2932) cell lines. As our findings from in silico analysis revealed potential binding sites for MYC on the SAMHD1 gene promoter, HL cell lines were treated with selective MYC inhibitors to assess possible MYC-associated regulation of SAMHD1 expression. The patient cohort included 212 cases of classical HL diagnosed and treated at the University of Texas MD Anderson Cancer Center (USA), University of Athens (Greece) and University of Crete (Greece). A control group of 4 reactive lymph nodes and 76 B-cell non-Hodgkin lymphomas (58 DLBCLs) was included in the study for comparison. All tissue samples were obtained prior to therapy. SAMHD1 expression was assessed by immunohistochemistry performed on TMAs with duplicate tumor cores from each case or full tissue sections using a monoclonal antibody (Bethyl Laboratories, San Antonio, TX). An arbitrary 10% cutoff was used to define SAMHD1 positivity in the neoplastic cells. Overall survival (OS) was defined as time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as time from diagnosis to relapse, death, or last follow-up. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models.Results: In 4 out of 5 HL cell lines, SAMHD1 protein levels were dramatically reduced as compared to all 3 DLBCL and other lymphoma and leukemia cell lines tested. Inhibition of MYC activity resulted in a substantial increase in the SAMHD1 protein level suggesting a possible MYC-associated gene regulation. Similarly, the neoplastic Hodgkin-Reed Sternberg (HRS) cells of classical HL were SAMHD1-negative in 132 of 212 (62.3%) patients. Among the SAMHD1-positive cases, the percentage of positive HRS cells ranged from 10 to 95% with a median 40%. By contrast, SAMHD1 was negative in only 15% of the control group of DLBCLs. In addition, most of the germinal center (GC) cells of the reactive lymph nodes were SAMHD1-positive with consistently higher intensity of immunostaining as compared to HRS cells of classical HL. No significant associations between SAMHD1 levels and clinicopathologic parameters were observed in the HL cohort. In a subset of 56 HL patients with long follow up period (median 195 months), FFS at 10 years was 79% vs. 71% for patients with SAMHD1-negative vs. SAMHD1-positive HL, respectively. Similarly, OS at 15 years was 71% vs. 63% for SAMHD1-negative vs. SAMHD1-positive HL, respectively. These differences did not reach statistical significance (p>0.05, log-rank) likely because of the relatively small number of patients included in this preliminary survival analysis.Conclusions: SAMHD1 is shown for the first time to be downregulated in HRS cells of classical HL further supporting its role as a tumor suppressor and its regulation may involve MYC. Preliminary survival analysis shows a statistical trend towards inferior FFS and OS for patients with SAMHD1-positive HRS cells, however, analysis of the entire cohort of HL patients is underway to draw definite conclusions. DisclosuresKhoury:Pfizer: Research Funding; Angle: Research Funding; Kiromics: Research Funding; Stemline Therapeutics: Research Funding.

The Tumor Suppressive mir-148a Is Epigenetically Inactivated in Classical Hodgkin Lymphoma.

DNA methylation was shown previously to be a crucial mechanism responsible for transcriptional deregulation in the pathogenesis of classical Hodgkin lymphoma (cHL). To identify epigenetically inactivated miRNAs in cHL, we have analyzed the set of miRNAs downregulated in cHL cell lines using bisulfite pyrosequencing. We focused on miRNAs with promoter regions located within or <1000 bp from a CpG island. Most promising candidate miRNAs were further studied in primary Hodgkin and Reed-Sternberg (HRS) cells obtained by laser capture microdissection. Last, to evaluate the function of identified miRNAs, we performed a luciferase reporter assay to confirm miRNA: mRNA interactions and therefore established cHL cell lines with stable overexpression of selected miRNAs for proliferation tests. We found a significant reverse correlation between DNA methylation and expression levels of mir-339-3p, mir-148a-3p, mir-148a-5p and mir-193a-5 demonstrating epigenetic regulation of these miRNAs in cHL cell lines. Moreover, we demonstrated direct interaction between miR-148a-3p and IL15 and HOMER1 transcripts as well as between mir-148a-5p and SUB1 and SERPINH1 transcripts. Furthermore, mir-148a overexpression resulted in reduced cell proliferation in the KM-H2 cell line. In summary, we report that mir-148a is a novel tumor suppressor inactivated in cHL and that epigenetic silencing of miRNAs is a common phenomenon in cHL.

Contribution of Epstein⁻Barr Virus Latent Proteins to the Pathogenesis of Classical Hodgkin Lymphoma.

Pathogenic viruses have evolved to manipulate the host cell utilising a variety of strategies including expression of viral proteins to hijack or mimic the activity of cellular functions. DNA tumour viruses often establish latent infection in which no new virions are produced, characterized by the expression of a restricted repertoire of so-called latent viral genes. These latent genes serve to remodel cellular functions to ensure survival of the virus within host cells, often for the lifetime of the infected individual. However, under certain circumstances, virus infection may contribute to transformation of the host cell; this event is not a usual outcome of infection. Here, we review how the Epstein–Barr virus (EBV), the prototypic oncogenic human virus, modulates host cell functions, with a focus on the role of the EBV latent genes in classical Hodgkin lymphoma.

Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma

AbstractDissecting the pathogenesis of classical Hodgkin lymphoma (cHL), a common cancer in young adults, remains challenging because of the rarity of tumor cells in involved tissues (usually <5%). Here, we analyzed the coding genome of cHL by microdissecting tumor and normal cells from 34 patient biopsies for a total of ∼50 000 singly isolated lymphoma cells. We uncovered several recurrently mutated genes, namely, STAT6 (32% of cases), GNA13 (24%), XPO1 (18%), and ITPKB (16%), and document the functional role of mutant STAT6 in sustaining tumor cell viability. Mutations of STAT6 genetically and functionally cooperated with disruption of SOCS1, a JAK-STAT pathway inhibitor, to promote cHL growth. Overall, 87% of cases showed dysregulation of the JAK-STAT pathway by genetic alterations in multiple genes (also including STAT3, STAT5B, JAK1, JAK2, and PTPN1), attesting to the pivotal role of this pathway in cHL pathogenesis and highlighting its potential as a new therapeutic target in this disease.

Distribution of 9p24 Abnormalities in Hematologic Malignancies with an Emphasis on Diffuse Large B-Cell Lymphoma

Introduction: 9p24 contains genes that are critical for immune evasion and propagating cell division. The loci for PD-L1, PD-L2, JAK2, and the histone demethylases KDM4C/JMJD2C are linked on 9p24 (Van Roosbroeck et al. Genes, Chromosomes and Cancer 2016). Amplification or rearrangements of this region have been described in the pathogenesis of classical Hodgkin lymphoma (cHL) and primary mediastinal large B-cell lymphoma (Ansell et al. New England Journal of Medicine 2015). Additionally, JAK2 amplification up-regulates PD-L1 and L2, which leads to increased T-cell inactivation and suggests synergy between these drug targets. The recent success of PD-1 blockade in numerous malignancies has led to the development and approval of PD-1 inhibitors in cHL as well as other cancers. Targeted therapies are approved for JAK2 inhibition, such as ruxolitinib, and are in development for histone demethylases, which illustrates the utility of identifying the 9p24 amplicon in hematologic malignancies (HM) (Van Roosbroeck et al. Genes, Chromsomes and Cancer 2016). The goal of this analysis is to better understand the distribution of 9p24 abnormalities across a broader range of leukemias and lymphomas in order to facilitate future studies of targeted therapy.Methods: The National Cancer Institute's Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer was queried for 9p24 breakpoint abnormalities within HM. Incidence of additions and rearrangements in chromosome 9p24 for all subtypes of HM listed in the Mitelman Database were calculated. Individual references were manually reviewed and pathologic data was extracted as available from the primary sources. Diffuse large B-cell lymphoma (DLBCL) cases were further assessed for co-incident rearrangement of MYC (8q24), BCL2 (18q21) and BCL6 (3q27) with 9p24. All subtypes with greater than 2% incidence of additions and/or rearrangements in chromosome 9p24 were reported.Results: 48,761 patients (pts) with HM across 74 lymphoid and myeloid subtypes were identified. 361 (<1%) pts had additions or rearrangements involving 9p24 across 53 subtypes of HM. 21 subtypes of HM had no 9p24 abnormalities. PTCL NOS had the highest incidence of aberrations (3.1% (6/193), 1.1% with additions and 2% with rearrangements) followed by cHL (2.7% (5/186), all additions) and DLBCL (2.7% (37/1413), 1.5% additions and 1.2% rearrangements) in malignancies with over 100 cases. Among DLBCL cases 9p24 was co-amplified/rearranged with MYC in 22% (8 cases), BCL6 27% (10), BCL2 14% (5) and in 3% (1) double-hit lymphomas. Minimal data was available on cell of origin; therefore, further analysis was not performed. No cases of DLBCL with 9p24 aberrations were T-cell rich DLBCL’s (0/37). Among the rarer lymphomas, extranodal NK/T cell lymphoma (11%, 2/18) and intravascular B cell lymphoma (7%, 1/14) had higher incidences of 9p24 additions or rearrangements (Table 1).Conclusion: Amplifications and rearrangement of 9p24 are rare in HM within the Mitelman database. Our study suggests that patients with DLBCL, adult T-cell leukemia/lymphoma, extranodal NK/T cell lymphoma, intravascular B cell lymphoma, peripheral T-cell lymphoma, and extranodal marginal zone B-cell may be considered for further studies with FISH and aCGH to further define the incidence of 9p24 alterations and potentially for targeted clinical trials. 9p24 abnormalities did not correlate with previously described phenotypic subtypes of DLBCL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutation analysis of tumor necrosis factor alpha-induced protein 3 gene in Hodgkin lymphoma

AimsSurvival and proliferation of Hodgkin and Reed-Sternberg (HRS) cells, the malignant cells of classical Hodgkin lymphoma (CHL), are dependent on constitutive activation of nuclear factor kB (NF-κB). A20, encoded by TNF alpha-induced protein 3 (TNFAIP3), one of the inhibitors of NF-kB, was found to be inactivated by deletions and/or point mutations in CHL. MethodsTNFAIP3 mutations were examined in 37 patients with CHL by using PCR and direct sequencing. In addition, protein expression of A20 was evaluated by immunohistochemistry. Epstein–Barr virus (EBV) status of HL samples was determined by EBV EBER chromogenic in situ hybridization (ISH). ResultsWe identified 8 mutation positive cases in a collective of 37 investigated cases (22%). Mutations were most frequent in the nodular sclerosis subtype. Our results revealed the tendency that cases harboring A20 mutations were negative for A20 staining. None of A20 mutation-positive CHL cases showed EBV infection. ConclusionsOur study confirms the involvement of the TNFAIP3 tumor suppressor gene in CHL. A20 may represent a suppressor of human lymphoma and provide a critical molecular link between chronic inflammation and cancer. None of A20 mutation-positive CHL cases showed EBV infection. This fact suggests complementing functions of TNFAIP3 inactivation and EBV infection in CHL pathogenesis and may represent an interesting point of further investigations.

The impact of EBV and HIV infection on the microenvironmental niche underlying Hodgkin lymphoma pathogenesis.

The pathogenesis of classical Hodgkin lymphoma (cHL) is still enigmatic, largely because its tumor cells, the so-called Hodgkin and Reed-Stenberg (HRS) cells, invariably reside in a prominent reactive microenvironment, are rare and therefore difficult to analyze. On the other hand, the broadly investigated cHL-derived cell lines are not unequivocally considered as suitable and representative models for this puzzling disease. Based on current knowledge, it appears that the cross talk between the tumor cells and the reactive infiltrate of the microenvironment is complex and that multiple mechanisms occur, making cHL a very heterogeneous disease. In 20-40% of cHL cases, HRS cells carry a monoclonal infection by Epstein Barr virus (EBV), which is considered a tumor-initiating factor. In these cases, EBV shows a latency type II infection pattern with the expression of latent membrane protein-1 (LMP-1), a viral oncoprotein that mimics CD40 activation. This scenario is particularly intriguing for the pathogenesis of cHL arising in HIV-infected patients, which, for still obscure reasons, is invariably EBV-associated with LMP-1 expression in HRS cells. Recent evidences are consistent with the occurrence of different pathogenic pathways variably triggered by virus infections (EBV and HIV), genetic alterations, and interactions with critical microenvironmental components. This review focuses on the different microenvironmental niches that characterize cHL of the general population as well as cases of HIV-infected patients. A more comprehensive understanding of the complex interplay existing between HRS and tumor microenvironment is pivotal for the development of more effective treatments, particularly for relapsed or refractory diseases.

Detection and prognostic value of recurrent exportin 1 mutations in tumor and cell-free circulating DNA of patients with classical Hodgkin lymphoma.

Classical Hodgkin lymphoma is one of the most common lymphomas and shares clinical and genetic features with primary mediastinal B-cell lymphoma. In this retrospective study, we analyzed the recurrent hotspot mutation of the exportin 1 (XPO1, p.E571K) gene, previously identified in primary mediastinal B-cell lymphoma, in biopsies and plasma circulating cell-free DNA from patients with classical Hodgkin lymphoma using a highly sensitive digital PCR technique. A total of 94 patients were included in the present study. This widely expressed XPO1 E571K mutation is present in one quarter of classical Hodgkin lymphoma patients (24.2%). Mutated and wild-type classical Hodgkin lymphomas were similar regarding the main clinical features. Patients with a detectable XPO1 mutation at the end of treatment displayed a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation in plasma cell-free DNA (2-year progression-free survival: 57.1%, 95% confidence interval: 30.1-100% versus 2-year progression-free survival: 90.5%, 95% confidence interval: 78.8-100%, respectively, P=0.0601). To conclude, the detection of the XPO1 E571K mutation in biopsy and plasma cell-free DNA by digital PCR may be used as a novel biomarker in classical Hodgkin lymphoma for both diagnosis and minimal residual disease, and pinpoints a crucial role of XPO1 in classical Hodgkin lymphoma pathogenesis. The detection of somatic mutation in the plasma cell-free DNA of patients represents a major technological advance in the context of liquid biopsies and noninvasive management of classical Hodgkin lymphoma.

Recurrent somatic loss of TNFRSF14 in classical Hodgkin lymphoma.

Investigation of the genetic lesions underlying classical Hodgkin lymphoma (CHL) has been challenging due to the rarity of Hodgkin and Reed-Sternberg (HRS) cells, the pathognomonic neoplastic cells of CHL. In an effort to catalog more comprehensively recurrent copy number alterations occurring during oncogenesis, we investigated somatic alterations involved in CHL using whole-genome sequencing-mediated copy number analysis of purified HRS cells. We performed low-coverage sequencing of small numbers of intact HRS cells and paired non-neoplastic B lymphocytes isolated by flow cytometric cell sorting from 19 primary cases, as well as two commonly used HRS-derived cell lines (KM-H2 and L1236). We found that HRS cells contain strikingly fewer copy number abnormalities than CHL cell lines. A subset of cases displayed nonintegral chromosomal copy number states, suggesting internal heterogeneity within the HRS cell population. Recurrent somatic copy number alterations involving known factors in CHL pathogenesis were identified (REL, the PD-1 pathway, and TNFAIP3). In eight cases (42%) we observed recurrent copy number loss of chr1:2,352,236-4,574,271, a region containing the candidate tumor suppressor TNFRSF14. Using flow cytometry, we demonstrated reduced TNFRSF14 expression in HRS cells from 5 of 22 additional cases (23%) and in two of three CHL cell lines. These studies suggest that TNFRSF14 dysregulation may contribute to the pathobiology of CHL in a subset of cases.

Macrophage Polarization Reflects T Cell Composition of Tumor Microenvironment in Pediatric Classical Hodgkin Lymphoma and Has Impact on Survival

Macrophages have been implicated in the pathogenesis of classical Hodgkin lymphoma (cHL) and have been suggested to have a negative impact on outcome. Most studies addressing the role of macrophages in cHL have relied on identification of macrophages by generic macrophage antigens, e.g., CD68. We have therefore conducted an in situ analysis of macrophage polarization in a series of 100 pediatric cHL (pcHL) cases using double staining immunohistochemistry, combining CD68 or CD163 with pSTAT1 (M1-like) or CMAF (M2-like). M1- or M2-polarised microenvironment was defined by an excess of one population over the other (>1.5). Expression of STAT1 and LYZ genes was also evaluated by RT-qPCR. Patients <14 years and EBV+ cases displayed higher numbers of CD68+pSTAT1+ cells than older children and EBV- cases, respectively (P=0.01 and P=0.02). A cytotoxic tumor microenvironment, defined by a CD8+/FOXP3+ ratio >1.5 was associated with higher numbers of CD68+pSTAT1+ (P=0.025) and CD163+pSTAT1+ macrophages (P<0.0005). Levels of STAT1 and LYZ expression were associated with the numbers of CD68+pSTAT1+ macrophages. EBV+ cHL cases disclosed a predominant M1 polarized microenvironment similar to Th1 mediated inflammatory disorders, while EBV- cHL showed a predominant M2 polarized microenvironment closer to Th2 mediated inflammatory diseases. Better overall-survival (OS) was observed in cases with higher numbers of CD163+pSTAT1+ macrophages (P=0.02) while larger numbers of CD163+CMAF+ macrophages were associated with worse progression-free survival (PFS) (P=0.02). Predominant M1-like polarization as disclosed by CD163+pSTAT1+/CD163+CMAF+ ratio > 1.5 was associated with better OS (P= 0.037). In conclusion, macrophage polarization in pcHL correlates with prevalent local T cell response and may be influenced by the EBV-status of neoplastic cells. Besides, M1-like and M2-like macrophages displayed differential effects on outcome in pcHL.

Expression of c-MYC and MMP9 in Alternatively Activated Macrophages (M2) of Classical Hodgkin Lymphoma

Classical Hodgkin Lymphoma (CHL) is a monoclonal lymphoid neoplasm that accounts for approximately 30% of all lymphomas in the United States. Although multiagent chemotherapy and radiation therapy have made CHL a highly curable disease, approximately 15-20% of patients are refractory to the treatment or relapsed after remission. The rich inflammatory cellular microenvironment plays an important role in the pathogenesis of CHL. Gene-expression profiling has identified a gene signature of tumor-associated macrophages (TAMs) that was significantly associated with primary treatment failure. The potential of macrophage-specific markers, CD68 and CD163, as prognostic markers for resistance to therapy in CHL patients has been investigated; however, these studies generated inconsistent results. Roughly TAMs could be characterized into two distinct polarization states; the classically activated type 1 macrophages (M1) associated with acute inflammation and T-cell immunity and the alternatively activated type 2 macrophages (M2) associated with the promotion of the tumor growth. In the present study, we hypothesize that the specific type of TAMs, M2, may predict poor prognosis in CHL. To test this hypothesis, we examined the correlation between expressions of two M2-specific markers, c-MYC and matrix metalloproteinase 9 (MMP-9), with disease recurrence in a total of 42 cases of CHL, mostly nodular sclerosis subtype. Both c-MYC and MMP-9 are good candidate markers because c-MYC has been shown to be a specific M2 marker and plays a central role in M2 differentiation. In addition, matrix metalloproteinase 9 (MMP-9) is a M2 specific marker that directly related to its protumoral function. Immunohistochemical staining showed that both c-MYC and MMP9 are expressed in a subset of macrophages, but not in Hodgkin Reed-Sternberg (HRS) cells. By comparing the percentage of positive stains of these two markers within HRS rich areas in the primary tumor samples, we found that there was no significant difference in either c-MYC or MMP9 staining between patients who later recurred (n=6) or did not recur (n=36). The role of TAMs or an unidentified subtype of TAMs in predicting resistance to therapy in CHL patients remains to be determined. DisclosuresNo relevant conflicts of interest to declare.

Cytokine gene polymorphisms and progression-free survival in classical Hodgkin lymphoma by EBV status: Results from two independent cohorts

BackgroundCytokines are important immune mediators of classical Hodgkin lymphoma (CHL) pathogenesis, and circulating levels at diagnosis may help predict prognosis. Germline single nucleotide polymorphisms (SNPs) in immune genes have been correlated with cytokine production and function. MethodsWe investigated whether selected germline SNPs in IL10 (rs1800890, rs1800896, rs1800871, rs1800872), TNFA (rs1800629), IL6 (rs1800795), ILRN (rs419598), INFG (rs2430561) and CCL17 (rs223828) were associated with circulating levels of related cytokines at diagnosis and progression-free survival (PFS) in CHL. Patients were from France (GELA, N=464; median age=32years) and the United States (Iowa/Mayo Specialized Program Of Research Excellence [SPORE], N=239; median age=38years); 22% of 346 CHL cases with EBV tumor status were positive. ResultsThere was no association with any of the SNPs with cytokine levels. Overall, there was no association of any of the SNPs with PFS. In exploratory analyses by EBV status, TNFA rs1800629 (HRAA/AG=2.41; 95%CI, 1.17–4.94) was associated with PFS in EBV-negative GELA patients, with similar trends in the SPORE patients (HRAA/AG=1.63; 95%CI, 0.61–4.40). In a meta-analysis of the two studies, TNFA (HRAA/AG=2.11; 95%CI, 1.18–3.77; P=0.01) was statistically significant, and further adjustment for the international prognostic system did not alter this result. ConclusionsThis study showed that germline variation in TNFA was associated with CHL prognosis for EBV-negative patients, which will require confirmation. These results support broader studies on the differential impact of genetic variation in immune genes on EBV-positive vs. EBV-negative CHL pathogenesis.

Analyzing primary Hodgkin and Reed-Sternberg cells to capture the molecular and cellular pathogenesis of classical Hodgkin lymphoma

The pathogenesis of classical Hodgkin lymphoma (cHL), the most common lymphoma in the young, is still enigmatic, largely because its Hodgkin and Reed-Sternberg (HRS) tumor cells are rare in the involved lymph node and therefore difficult to analyze. Here, by overcoming this technical challenge and performing, for the first time, a genome-wide transcriptional analysis of microdissected HRS cells compared with other B-cell lymphomas, cHL lines, and normal B-cell subsets, we show that they differ extensively from the usually studied cHL cell lines, that the lost B-cell identity of cHLs is not linked to the acquisition of a plasma cell-like gene expression program, and that Epstein-Barr virus infection of HRS cells has a minor transcriptional influence on the established cHL clone. Moreover, although cHL appears a distinct lymphoma entity overall, HRS cells of its histologic subtypes diverged in their similarity to other related lymphomas. Unexpectedly, we identified 2 molecular subgroups of cHL associated with differential strengths of the transcription factor activity of the NOTCH1, MYC, and IRF4 proto-oncogenes. Finally, HRS cells display deregulated expression of several genes potentially highly relevant to lymphoma pathogenesis, including silencing of the apoptosis-inducer BIK and of INPP5D, an inhibitor of the PI3K-driven oncogenic pathway.

HLA associations in classical Hodgkin lymphoma: EBV status matters.

The pathogenesis of classical Hodgkin lymphoma (cHL) involves environmental and genetic factors. To explore the role of the human leukocyte antigen (HLA) genes, we performed a case-control genotyping study in 338 Dutch cHL patients using a PCR-based sequence-specific oligonucleotide probe (SSOP) hybridization approach. The allele frequencies were compared to HLA typings of more than 6,000 controls. The age of the cHL patients varied between 13 and 81 years with a median of 35 years. Nodular sclerosis subtype was the most common subtype (87%) and EBV was detected in 25% of the cHL patients. HLA-B5 was significantly increased and HLA-DR7 significantly decreased in the total cHL patient population as compared to controls. Two class II associations were observed to be specific for the EBV− cHL population with an increase of HLA-DR2 and HLA-DR5. Allele frequencies of HLA-A1, HLA-B37 and HLA-DR10 were significantly increased in the EBV+ cHL population; these alleles are in strong linkage disequilibrium and form a common haplotype in Caucasians. The allele frequency of HLA-A2 was significantly decreased in the EBV+ cHL population. Analysis of haplotypes with a frequency of >1% revealed a significant increase of HLA-A2-B7-DR2 in EBV− cHL as compared to controls. SSOP association analysis revealed significant differences between EBV+ and EBV− cHL patients for 19 probes that discriminate between HLA-A*01 and HLA-A*02. In conclusion, the HLA-A1 and HLA-A2 antigens and not specific single nucleotide variants shared by multiple alleles are responsible for the association with EBV+ cHL. Furthermore several new protective and predisposing HLA class I and II associations for the EBV+, the EBV− and the entire cHL population were identified.

Huang X, Kushekhar K, Nolte I, Kooistra W, Visser L, Bouwman I, Kouprie N, Veenstra R, van Imhoff G, Olver B, Houlston RS, Poppema S, Diepstra A, Hepkema B, van den Berg A. Multiple HLA class I and II associations in classical Hodgkin lymphoma and EBV status defined subgroups. Blood. 2011;118(19):5211–5217.

The pathogenesis of classical Hodgkin lymphoma (cHL) involves environmental and genetic factors. To explore the role of the human leukocyte antigen (HLA) genes, we performed a case-control genotyping study in 338 Dutch cHL patients and more than 5000 controls using a PCR-based sequence-specific oligonucleotide probe hybridization approach. HLA-A68 and HLA-DR11 (5) were significantly increased in the cHL patient population compared with the controls. Three class II associations were observed in the EBV- cHL population with an increase of HLA-DR15 (2) and a decrease of HLA-DR4 and HLA-DR7. Allele frequencies of HLA-A1, HLA-B37, and HLA-DR10 were significantly increased in the EBV+ cHL population; these alleles are in strong linkage disequilibrium and form a common haplotype in whites. The allele frequency of HLA-A2 was significantly decreased in the EBV- cHL population. Sequence-specific oligonucleotide probe analysis revealed significant differences between EBV+ and EBV- cHL patients for 19 probes that discriminate between HLA-A*01 and HLA-A*02. In conclusion, the HLA-A1 and HLA-A2 antigens and not specific single nucleotide variants shared by multiple alleles are responsible for the association with EBV+ cHL. Furthermore, several new protective and predisposing HLA class I and II associations for the EBV+, the EBV-, and the entire cHL population were identified. (Blood. 2011;118(19):5211-5217)

Multiple HLA class I and II associations in classical Hodgkin lymphoma and EBV status defined subgroups

AbstractThe pathogenesis of classical Hodgkin lymphoma (cHL) involves environmental and genetic factors. To explore the role of the human leukocyte antigen (HLA) genes, we performed a case-control genotyping study in 338 Dutch cHL patients and more than 5000 controls using a PCR-based sequence-specific oligonucleotide probe hybridization approach. HLA-A68 and HLA-DR11 (5) were significantly increased in the cHL patient population compared with the controls. Three class II associations were observed in the EBV− cHL population with an increase of HLA-DR15 (2) and a decrease of HLA-DR4 and HLA-DR7. Allele frequencies of HLA-A1, HLA-B37, and HLA-DR10 were significantly increased in the EBV+ cHL population; these alleles are in strong linkage disequilibrium and form a common haplotype in whites. The allele frequency of HLA-A2 was significantly decreased in the EBV+ cHL population. Sequence-specific oligonucleotide probe analysis revealed significant differences between EBV+ and EBV− cHL patients for 19 probes that discriminate between HLA-A*01 and HLA-A*02. In conclusion, the HLA-A1 and HLA-A2 antigens and not specific single nucleotide variants shared by multiple alleles are responsible for the association with EBV+ cHL. Furthermore, several new protective and predisposing HLA class I and II associations for the EBV+, the EBV−, and the entire cHL population were identified.