Pathogenesis Of 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.

Nivolumab-AVD Is Better Tolerated and Improves Progression-Free Survival Compared to Bv-AVD in Older Patients (Aged ≥60 Years) with Advanced Stage Hodgkin Lymphoma Enrolled on SWOG S1826

Background: Survival rates in patients (pts) with classic Hodgkin lymphoma (HL) aged ≥60 years (y) are lower than younger pts. The difference is due to comorbidities, impaired performance status, decreased chemotherapy tolerance, and increased treatment-related toxicity and mortality. The frontline regimen Bv-AVD can be challenging to administer in older patients due to toxicities, particularly infection and neuropathy. The PD-1 pathway is central to pathogenesis of HL; PD-1 blockade is effective in HL. The randomized, phase 3 trial, S1826, was conducted by the National Clinical Trials Network to evaluate N-AVD vs Bv-AVD in pts with newly diagnosed advanced stage (AS) HL and enrolled 994 pts; 97 were eligible and aged ≥60y. Methods: In this subset analysis, eligible pts were ≥60y with stage 3-4 HL. Pts were randomized 1:1 to 6 cycles of N-AVD or Bv-AVD. G-CSF neutropenia prophylaxis was optional with N-AVD and required with Bv-AVD. Pre-specified pts could receive radiation therapy (RT) to residual lesions on end of treatment PET. Pts were stratified by age, international prognostic score (IPS), and intent to use RT. Response and progression were assessed by investigators using 2014 Lugano Classification. The primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS), event-free survival (EFS), and detailed toxicity and safety events. EFS events were death without progression, progression/relapse, receipt of non-protocol systemic anti-lymphoma therapy without progression, or receipt of RT in a pt not declared eligible for RT at registration or who did not meet protocol-specified criteria for RT. Results: 97 pts aged ≥60y were enrolled from 7/9/19-10/5/22 and were randomized to N-AVD (n=48) or Bv-AVD (n=49). Median age was 66y (range, 60-83y), 62% were male, 86% were white, 3% were black, and 10% were Hispanic. 61% had stage IV disease and 47% had IPS 4-7. 69% of pts on N-AVD and 92% of pts on Bv-AVD received any G-CSF. No pts received RT per protocol. 95 pts were evaluable for safety analysis. While grade (gr) ≥3 hematologic toxicity occurred in 52% of pts on N-AVD and 38% on Bv-AVD (gr 3 neutropenia 48% vs 30%), febrile neutropenia, sepsis, and infections were lower for pts who received N-AVD vs Bv-AVD. (Table) Peripheral neuropathy was much less frequent with N-AVD than Bv-AVD in overall incidence (sensory: 31% vs 66% and motor: 8% vs 15%, respectively) and severity (grades ≥2, sensory: 10% vs 49% and motor: 0% vs 8%, respectively). (Table) The following adverse events, AEs (mainly <gr 3) were less frequent on N-AVD vs Bv-AVD: nausea (38% vs 62%), diarrhea (25% vs 45%), anorexia (15% vs 38%) and weight loss (4% vs 36%). Hypothyroidism (15% vs 0%), and rash (16% vs 2%, no gr ≥3) were more frequent on N-AVD than Bv-AVD; rates of other immune-related toxicities were similar between arms. In this subset analysis, median follow-up 12.1 months, PFS was superior in the N-AVD arm [HR 0.35 (95% CI 0.12-1.02) stratified one-sided logrank p=0.022]; 1-yr PFS was 93% (95% CI 79-98%) for N-AVD and 64% (95% CI 45-77%) for Bv-AVD. (Figure) 1-yr EFS was 93% (95% CI 79-98%) for N-AVD and 57% for Bv-AVD (95% CI 38-72%) [HR 0.19 (95% CI 0.06-0.61) stratified one-sided logrank p=0.0011]. 1-yr OS was 95% (95% CI 83-99%) for N-AVD and 83% (95% CI 67-92%) for Bv-AVD [HR 0.35 (95% CI 0.07-1.75) stratified one-sided logrank p=0.091]. On N-AVD, there were 2 deaths (due to infection/sepsis) and 3 progressions/relapses; on Bv-AVD, there were 7 deaths (5 due to infection/sepsis, 1 to pneumonitis, 1 unknown) and 8 progressions/relapses. Non-relapse mortality was 4% for N-AVD vs 14% for Bv-AVD. Treatment was discontinued early in 5 pts (10%) on N-AVD and 16 pts (33%) on Bv-AVD. N was discontinued early in 7 pts (15%) and Bv in 19 pts (39%). Reasons for discontinuation included (N vs Bv): AE (5 vs 14 pts), death (1 vs 3 pts), and disease progression (0 vs 1 pt). Conclusions: N-AVD improved PFS and EFS, and was better tolerated than Bv-AVD in pts aged ≥60 with AS HL. Substantially more pts discontinued Bv-AVD than N-AVD, primarily due to toxicity. Fewer deaths occurred on N-AVD than Bv-AVD. N-AVD is poised to become a standard of care for older AS HL pts fit for anthracycline-based combination therapy. Funding provided by the National Cancer Institute of the National Institutes of Health CA180888, CA180819, CA180821, CA180820, CA180863, CA189955 and funding/drug provided by BMS, drug provided by SeaGen for pts enrolled in Canada.

Analysis of the association between Epstein Barr virus infection and Hodgkin lymphoma

Epstein Barr virus plays an important role in the pathogenesis of Hodgkin lymphoma. However, the frequency of its association in Bangladeshi people has not been widely studied. The aim of this study was to determine the association of Epstein Barr virus association in Hodgkin Lymphoma through detection of Latent Membrane Protein 1 by immunohistochemistry and serum Epstein Barr virus viral capsid antigen IgG antibody titer by serology. This was a cross-sectional study in purposively selected 45 histologically diagnosed cases of Hodgkin lymphoma at Bangabandhu Sheikh Mujib Medical University, Dhaka, during the period of March 2017 to December 2018. Latent membrane protein 1 was positive in 71.1% of cases of Hodgkin lymphoma. Among these positive cases, 96% of cases had significantly raised titer of serum Epstein Barr virus viral capsid antigen IgG antibody (P<0.0001 obtained by Fisher’s Exact test), which had a male predominance. Mixed cellularity classical Hodgkin lymphoma showed the highest positivity. Bangabandhu Sheikh Mujib Medical University Journal 2022;15(4):32-36

P037: High breadth whole exome sequencing of circulating tumor DNA identifies novel recurrent genetic alterations in Hodgkin lymphoma

Hodgkin lymphoma (HL) is a B-cell lymphoma with a generally favorable prognosis. However, side effects of aggressive front-line treatment and difficult-to-treat relapsed/refractory disease are still a clinical challenge. A better understanding of the biological diversity of HL at baseline might help to further individualize the treatment of patients and thus ultimately improve outcome. A broad characterization of the genetic drivers of HL is highly important to improve our understanding of the biological diversity of HL. Here, we present the results of our study using whole-exome sequencing of circulating tumor (ct)DNA to broadly characterize the HL oncogenome in 165 patients. After extraction of germline and cell-free DNA, WES was performed, targeting 37 Mb of the human genome. An in-house customized bioinformatic pipeline including twofold error reduction using unique molecular identifiers (UMIs) and digital error suppression was used as previously described by us (Sobesky et. al., Med, 2021). The median ctDNA concentration per patient was 2.26 log haploid genome equivalents per milliliter (log hGE/ml) (range: 1.41–4.18 log hGE/ml) without significant correlation with clinical parameters such as sex, international prognostic score (IPS) or clinical stage. In addition to the detection of several genetic aberrations involved in the pathogenesis of HL (such as B2M, CSF2RB, GNA13, SOCS1), we were able to detect several novel, recurrently mutated potential oncogenes and tumor suppressor genes. Among the most frequently mutated genes not previously described in HL were olfactory receptor family 10 subfamily G member 7 (OR10G7) (11.5%), Apolipoprotein B (APOB) (10.3%) and Filaggrin 2 (FLG2) (9.7%). Furthermore, several previously undescribed copy number variations (CNV) were detected (losses: 13q13.1, 1p36.13a, 6p21.33; gains: 19q13.42, 9p24, 12q12). To decipher the detected genes’ and CNVs’ contribution to the biological diversity of HL, we performed non-negative matrix factorization. Furthermore, neoantigen load was assessed through human-leukocyte antigen (HLA) sequencing. Detailed results will be presented at the meeting. In conclusion, our ctDNA based WES approach provides sufficient sensitivity to both perform genotyping in HL and detecting novel genomic aberrations potentially involved in the pathogenesis of HL.

EVOLUTION OF DRUG THERAPY FOR CLASSICAL HODGKIN LYMPHOMA

Hodgkin's lymphoma is a malignant disease of the lymphatic system. Hodgkin's lymphoma was first described by Dr. Thomas Hodgkin in 1832 and later named “Hodgkin's disease” by Samuel Wilkes. Hodgkin's lymphoma accounts for about 24 % of all lymphomas. Hodgkin's lymphoma is classified as classical and nodular lymphoid-predominant (Nodular type of lymphoidpredominant Hodgkin's lymphoma). Classical Hodgkin's lymphoma includes the following histologic variants: nodular sclerosis variant (types I and II), mixed cell variant, classic lymphocyte-rich variant, and rare lymphoid depletion variant. Epidemiological and serological studies showed the involvement of the Epstein-Barr virus into Hodgkin's lymphoma etiology, since its genome was found in the study of the biopsy material samples from patients with Hodgkin's lymphoma. A relationship with the human immunodeficiency virus (HIV) was revealed as well, and patients infected with HIV have a significantly increased risk of developing Hodgkin's lymphoma compared to healthy people. An in-depth study of the Hodgkin's lymphoma pathophysiology revealed new therapeutic targets in the treatment of this disease. All these discoveries changed the understanding of the Hodgkin's lymphoma pathogenesis, and were important for the development of new methods of treatment. The history of therapy begins on the cusp of the 19th and 20th centuries. Over the past four decades, achievements in radiation therapy and combined chemotherapy have significantly improved overall survival of patients with Hodgkin's lymphoma. Currently, more than 80 % of patients under 60 years old with first diagnosed Hodgkin's lymphoma can be cured from this disease after first-line chemotherapy.

Hodgkin Lymphoma in People Living with HIV.

Simple SummaryHodgkin lymphoma (HL) is a non-AIDS defining neoplasm, but people living with HIV (PLWH) have between a 5- and 26-fold higher risk of developing it than the general population. Epstein-Barr virus is present in almost all HIV-related HL cases, and plays an important role in its etiopathogenesis. Despite the aggressive characteristics, the prognosis of HL affecting PLWH is similar to that of the general population if patients are treated following the same recommendations. Administration of cART concomitantly with chemotherapy is highly recommended. However, this combination may be challenging due to drug–drug interactions and overlapping toxicity. Thus, interdisciplinary collaboration between hemato-oncologists and HIV specialists is crucial for the optimal treatment of both lymphoma and HIV infection.Despite widespread use of combined antiretroviral therapy (cART) and increased life expectancy in people living with HIV (PLWH), HIV-related lymphomas (HRL) remain a leading cause of cancer morbidity and mortality for PLWH, even in patients optimally treated with cART. While the incidence of aggressive forms of non-Hodgkin lymphoma decreased after the advent of cART, incidence of Hodgkin lymphoma (HL) has increased among PLWH in recent decades. The coinfection of Epstein–Barr virus plays a crucial role in the pathogenesis of HL in the HIV setting. Currently, PLWH with HRL, including HL, are treated similarly to HIV-negative patients and, importantly, the prognosis of HL in PLWH is approaching that of the general population. In this regard, effective cART during chemotherapy is strongly recommended since it has been shown to improve survival rates in all lymphoma subtypes, including HL. As a consequence, interdisciplinary collaboration between HIV specialists and hemato-oncologists for the management of potential drug–drug interactions and overlapping toxicities between antiretroviral and antineoplastic drugs is crucial for the optimal treatment of PLWH with HL. In this article the authors review and update the epidemiological, clinical and biological aspects of HL presenting in PLWH with special emphasis on advances in prognosis and the factors that have contributed to it.

Interaction between ERAP Alleles and HLA Class I Types Support a Role of Antigen Presentation in Hodgkin Lymphoma Development.

Simple SummaryHodgkin lymphoma (HL) is a common lymphoma in young adults derived from B cells. Emerging evidence suggests that antigen presentation by the malignant B cells is critically involved in HL pathogenesis. In fact, genetic variants of the antigen presenting Human Leukocyte Antigens (HLA) are strongly associated with HL susceptibility. Interestingly, the endoplasmic reticulum aminopeptidase (ERAP)1 and ERAP2 genes, that code for enzymes that process antigens, also appear to be associated. In this study, we show that genetic variants of ERAP genes strongly affect expression levels of ERAP1 and ERAP2. In addition, we find that certain ERAP variants interact with specific HLA class I types in HL patients. This suggests that mechanisms that determine the repertoire of antigens that are presented to the immune system, affect the chance of developing HL. Our findings therefore support a prominent role of antigen presentation in HL susceptibility.Genetic variants in the HLA region are the strongest risk factors for developing Hodgkin lymphoma (HL), suggesting an important role for antigen presentation. This is supported by another HL-associated genomic region which contains the loci of two enzymes that process endogenous proteins to peptides to be presented by HLA class I, i.e., endoplasmic reticulum aminopeptidase 1 (ERAP1) and ERAP2. We hypothesized that ERAP and HLA class I type interact in HL susceptibility, as shown previously for several autoimmune diseases. We detected ERAP1 and ERAP2 expression in tumor cells and cells in the microenvironment in primary HL tissue samples. Seven ERAP SNPs and ERAP1 haplotypes showed strong associations with RNA and protein levels of ERAP1 and ERAP2 in LCLs and HL cell lines. Analysis of HLA class I types, ERAP SNPs and ERAP haplotypes by direct genotyping or imputation from genome-wide association data in 390 HL patients revealed significant interactions between HLA-A11, rs27038 and the rs27038 associated ERAP haplotype, as well as between HLA-Cw2 and rs26618. In conclusion, our results show that ERAP and HLA class I interact in genetic susceptibility to HL, providing further evidence that antigen presentation is an important process in HL susceptibility and pathogenesis.

Hodgkin Lymphoma Monozygotic Triplets Reveal Divergences in DNA Methylation Signatures.

We studied DNA methylation profiles in four different cell populations from a unique constellation of monozygotic triplets in whom two had developed Hodgkin Lymphoma (HL). We detected shared differences in DNA methylation signatures when comparing the two HL-affected triplets with the non-affected triplet. The differences were observed in naïve B-cells and marginal zone-like B-cells. DNA methylation differences were also detected when comparing each of the HL-affected triplets against each other. Even though we cannot determine whether treatment and/or disease triggered the observed differences, we believe our data are important on behalf of forthcoming studies, and that it might provide important clues for a better understanding of HL pathogenesis.

SWOG S1826: A Phase III, Randomized Study of Nivolumab Plus AVD or Brentuximab Vedotin Plus AVD in Patients with Newly Diagnosed Advanced Stage Classical Hodgkin Lymphoma

SWOG S1826: A Phase III, Randomized Study of Nivolumab Plus AVD or Brentuximab Vedotin Plus AVD in Patients with Newly Diagnosed Advanced Stage Classical Hodgkin Lymphoma

Essential role of the linear ubiquitin chain assembly complex and TAK1 kinase in A20 mutant Hodgkin lymphoma

More than 70% of Epstein-Barr virus (EBV)-negative Hodgkin lymphoma (HL) cases display inactivation of TNFAIP3 (A20), a ubiquitin-editing protein that regulates nonproteolytic protein ubiquitination, indicating the significance of protein ubiquitination in HL pathogenesis. However, the precise mechanistic roles of A20 and the ubiquitination system remain largely unknown in this disease. Here, we performed high-throughput CRISPR screening using a ubiquitin regulator-focused single-guide RNA library in HL lines carrying either wild-type or mutant A20. Our CRISPR screening highlights the essential oncogenic role of the linear ubiquitin chain assembly complex (LUBAC) in HL lines, which overlaps with A20 inactivation status. Mechanistically, LUBAC promotes IKK/NF-κB activity and NEMO linear ubiquitination in A20 mutant HL cells, which is required for prosurvival genes and immunosuppressive molecule expression. As a tumor suppressor, A20 directly inhibits IKK activation and HL cell survival via its C-terminal linear-ubiquitin binding ZF7. Clinically, LUBAC activity is consistently elevated in most primary HL cases, and this is correlated with high NF-κB activity and low A20 expression. To further understand the complete mechanism of NF-κB activation in A20 mutant HL, we performed a specifically designed CD83-based NF-κB CRISPR screen which led us to identify TAK1 kinase as a major mediator for NF-κB activation in cells dependent on LUBAC, where the LUBAC-A20 axis regulates TAK1 and IKK complex formation. Finally, TAK1 inhibitor Takinib shows promising activity against HL in vitro and in a xenograft mouse model. Altogether, these findings provide strong support that targeting LUBAC or TAK1 could be attractive therapeutic strategies in A20 mutant HL.

Molecular Pathogenesis of Hodgkin Lymphoma: Past, Present, Future.

Our understanding of the tumorigenesis of classical Hodgkin lymphoma (cHL) and the formation of Reed–Sternberg cells (RS-cells) has evolved drastically in the last decades. More recently, a better characterization of the signaling pathways and the cellular interactions at play have paved the way for new targeted therapy in the hopes of improving outcomes. However, important gaps in knowledge remain that may hold the key for significant changes of paradigm in this lymphoma. Here, we discuss the past, present, and future of cHL, and review in detail the more recent discoveries pertaining to genetic instability, anti-apoptotic signaling pathways, the tumoral microenvironment, and host-immune system evasion in cHL.

Comprehensive Cell Specific Transcriptome Profiling of a Pediatric Hodgkin Lymphoma Cohort

Introduction: Pathogenic Hodgkin Reed-Sternberg (HRS) cells constitute approximately 1% of Hodgkin lymphoma (HL) tumor cells. Studies characterizing genomic lesions and gene expression of HRS gene cells have been limited due to technical challenges of studying these rare cells, and the majority of existing data has focused on adult HL. We therefore developed a multi-parameter flow sorting strategy to isolate viable cells from pediatric HL tumors and to define the transcriptomes of HRS cells and infiltrating lymphocytes in order to inform underlying mechanisms of HL pathogenesis and also create an opportunity to identify cell-specific biomarkers to predict disease risk and response to therapy. Methods : Flow cytometry was used to sort HRS cells, CD4+ T cells, CD8+ T cells, and CD20+/30+B cells from pediatric subjects' HL lesions and control tonsils. Purity was confirmed by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Affymetrix GeneChip HTA 2.0 was used to assess the gene expression profiles (GEPs) for 16 HRS primary tumor cell samples, 14 HL CD4+ and CD8+ T cell samples, 6 control tonsillar CD20+, CD30+, CD4+, and CD8+ cell samples, and 6 HL cell lines. Unsupervised hierarchical clustering and principal component analysis (PCA) were used to determine relatedness, and Cibersort was performed to confirm the phenotype of the sorted cell types. GEPs of HRS, HL CD4+, and HL CD8+ cells were compared to respective controls using a univariate t-test. Significance was determined using a multivariate permutation test with the confidence level of FDR assessment at 80 percent and the maximum allowed proportion of false-positive proteins at 0.1. Gene set enrichment analysis (GSEA) and ingenuity pathway analysis (IPA) were performed to analyze DEGs. Results: Effectiveness of the sorting strategy of HRS cells was confirmed by quantitative RT-PCR and IHC that demonstrated significant enrichment of CD30expression and CD30+ cells in the sorted HRS cell fraction. GEP comparisons were performed for 13 HL samples with matched HRS/CD4+/CD8+ cells: HRS vs. control tonsil CD20+/CD30+ (1934 and 3846 DEGs, respectively), HL CD4+ vs. control CD4+ (635 DEGs), HL CD8+ vs. control CD8+ (2 DEGs). We carried out a transcriptomic analysis of HRS cells, and a set of multifunctional genes were more than 2-fold downregulated (P < .001), involved in telomere maintenance and packaging (TERF2, RFC3, DNA2 and a group of HIST1) when compared to healthy lymph node CD30+ cells. A set of genes related to cytokine/chemokine dysregulation was also upregulated in HRS cells, including IL6, CCL18, and CXCL9. IPA and GSEA of specific HRS genes were also performed and demonstrated pathways associated with HL pathogenesis, including NFĸB activation and T cell exhaustion. Over-expression of genes associated with T cell pathways was demonstrated in HRS cells. While this may be a result of T cell rosetting and contamination, it may also reflect innate T cell signature within HRS cells, as HRS cells clustered separately from T cells in both unsupervised hierarchical clustering and PCA. Cibersort analysis of HRS cells revealed a heterogeneous phenotype that may reflect aberrant differentiation. In comparing clinical characteristics within HRS cells, TCEAL1 was elevated in slow vs. rapid early responders and 3 DEGs were identified when comparing EBV+/- samples. Within HL CD8 cells, KLF2 was elevated in EBV- samples. Conclusions: This study was the first to successfully isolate highly purified HRS cell populations from whole HL lesions in a pediatric HL cohort. Transcriptomic analysis of pediatric HRS cells identified mechanisms previously associated with HL pathogenesis, and also identified potential novel mechanisms, including telomere maintenance. Additional analyses demonstrated significant heterogeneity of HRS trasncriptomes across specimens that may reflect distinct differentiation pathways and differences in HRS-immune cell interactions. Finally, this study identified increased expression of some genes associated with EBV status and response to therapy. Future studies in an expanded cohort will validate these findings, compare pediatric and adult GEPs, and test these cell-specific biomarkers into the current risk stratification strategies of prospective clinical trials. Disclosures No relevant conflicts of interest to declare.

Genomic Analysis of Hodgkin Lymphoma

The genetic analysis of the Hodgkin and Reed/Sternberg (HRS) tumor cells in Hodgkin lymphoma (HL) is very much hampered by the rarity of these cells, typically accounting for only about 1% of cells in the lymphoma tissue. Moreover, only very few cell lines were established from HL, partly with an uncertain origin. Thus, for the identification of somatic mutations in HRS cells, methods had to be established to isolate the rare HRS cells by microdissection from tissue sections or by cell sorting from cell suspensions. Initial studies were focussed on candidate gene approaches, often inspired by results from the prior analysis of HL cell lines. These early studies revealed numerous recurrent mutations in members of the NF-κB and JAK/STAT pathways, causing or at least contributing to the constitutive activation of these signaling pathways.1 The pathobiological relevance of the constitutive activation of NF-κB and JAK/STAT factors was validated by functional studies with HL cell lines. A first whole exome sequencing analysis of flow-sorted HRS cells was published by Reichel and colleagues.2 B2M was found to be inactivated by somatic mutations in 7/10 cases, and caused loss of major histocompatibility complex 1 expression. Although later studies with larger case series identified such mutations in only about 20-30% of cases,3,4 B2M inactivation is nevertheless an important factor for immune evasion of HRS cells and a frequently mutated gene in HL. A second exome sequencing study of HRS cells used microdissected tumor cells and revealed as a major novel finding frequent somatic mutations in the STAT6 gene (ca. 30% of cases).3 The genetic lesions in STAT6 are gain-of-function mutations. Overall nearly 90% of HL cases show genetic alterations in members of the JAK/STAT pathway, further demonstrating the major role of this pathway in HL pathogenesis. Our group is currently performing studies of microdissected HRS cells by whole genome sequencing, aiming to identify further genetic lesions in HRS cells beyond those affecting exons. An alternative attractive approach to uncover mutations in HRS cells is the genetic analysis of circulating DNA in plasma, because HRS cell-derived DNA fragments can be identified in most patients at the time of diagnosis. A targeted sequencing study of circulating tumor DNA showed the feasibility of this approach and validated, for example, the frequent occurrence of STAT6 mutations in HL. In rare instances, a HL can co-occur with another lymphoma in the same patient. Such composite lymphomas frequently show a common clonal origin of both lymphomas from a mature B cell.5 Composite lymphomas are therefore elegant models to study the multi-step transformation process in lymphomagenesis. We are currently performing a whole exome sequencing analysis of composite classical HL and B-cell Non-Hodgkin lymphomas, to reveal the pattern of early shared mutations and later distinct mutations. In each of the cases under investigations, such shared and distinct mutations were indeed identified. Küppers R, Engert A, Hansmann ML. Hodgkin lymphoma. J Clin Invest. 2012; 122: 3439. Reichel J, Chadburn A, Rubinstein PG, et al. Flow sorting and exome sequencing reveal the oncogenome of primary Hodgkin and Reed-Sternberg cells. Blood. 2015; 125: 1061. Tiacci E, Ladewig E, Schiavoni G, et al. Pervasive mutations of JAK-STAT pathway genes in classical Hodgkin lymphoma. Blood. 2018; 131: 2454. Spina V, Bruscaggin A, Cuccaro A, et al. Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkinlymphoma. Blood. 2018; 131: 2413. Küppers R, Dührsen U, Hansmann ML. Pathogenesis, diagnosis, and treatment of composite lymphomas. Lancet Oncol. 2014; 15: e435. Disclosures No relevant conflicts of interest to declare.

An intronic deletion in megakaryoblastic leukemia 1 is associated with hyperproliferation of B cells in triplets with Hodgkin lymphoma.

Megakaryoblastic leukemia 1 (MKL1) is a coactivator of serum response factor and together they regulate transcription of actin cytoskeleton genes. MKL1 is associated with hematologic malignancies and immunodeficiency, but its role in B cells is unexplored. Here we examined B cells from monozygotic triplets with an intronic deletion in MKL1, two of whom had been previously treated for Hodgkin lymphoma (HL). To investigate MKL1 and B-cell responses in the pathogenesis of HL, we generated Epstein-Barr virus-transformed lymphoblastoid cell lines from the triplets and two controls. While cells from the patients with treated HL had a phenotype close to that of the healthy controls, cells from the undiagnosed triplet had increased MKL1 mRNA, increased MKL1 protein, and elevated expression of MKL1-dependent genes. This profile was associated with elevated actin content, increased cell spreading, decreased expression of CD11a integrin molecules, and delayed aggregation. Moreover, cells from the undiagnosed triplet proliferated faster, displayed a higher proportion of cells with hyperploidy, and formed large tumors in vivo. This phenotype was reversible by inhibiting MKL1 activity. Interestingly, cells from the triplet treated for HL in 1985 contained two subpopulations: one with high expression of CD11a that behaved like control cells and the other with low expression of CD11a that formed large tumors in vivo similar to cells from the undiagnosed triplet. This implies that pre-malignant cells had re-emerged a long time after treatment. Together, these data suggest that dysregulated MKL1 activity participates in B-cell transformation and the pathogenesis of HL.

Classical Hodgkin's Lymphoma in the Era of Immune Checkpoint Inhibition.

The ligation of programmed cell death 1 (PD-1) with programmed cell death ligand PD-L activates the immune checkpoint leading to T-cell dysfunction, exhaustion, and tolerance, especially in Hodgkin lymphoma (HL) where the PD-L/ Janus kinase (Jak) signaling was frequently found altered. Anti-PD-1 or anti-PD-L1 monoclonal antibodies can reverse this immune checkpoint, releasing the brake on T-cell responses. The characterization of the mechanisms regulating both the expression of PD-1 and PD-L and their function(s) in HL is ongoing. We provide in this review the recent findings focused on this aim with special attention on the major research topics, such as adverse events and resistance to PD-1–PD-L1 inhibitor treatment, together with a part about angiogenesis, extracellular vesicles, and microbiome in HL pathogenesis.

Epstein–Barr virus-encoded LMP1 induces ectopic CD137 expression on Hodgkin and Reed–Sternberg cells via the PI3K-AKT-mTOR pathway

CD137 is a potent co-stimulatory molecule on activated T cells, and its ligand (CD137L) is expressed on antigen presenting cells (APC). Ectopic expression of CD137 has been identified on Hodgkin Reed–Sternberg (HRS) cells, the malignant cells in Hodgkin Lymphoma (HL), and CD137 on HRS cells was found to support growth of HRS cells and escape from immune surveillance. HRS cells are mostly derived from B cells, which poses the question of how B cells acquire ectopic CD137 expression during the transformation process. HL is associated with Epstein–Barr virus (EBV) infection. We show that the EBV latent membrane protein 1 (LMP1) induces expression of CD137 in HRS cell lines. In a HL tissue microarray, 96% of the CD137-positive HL cases stained positive for LMP1. LMP1 utilizes the PI3K-AKT-mTOR pathway for inducing CD137 expression. These findings support the role of EBV in HL pathogenesis.

How Epstein-Barr Virus “Manipulates” The Tumoral Microenvironment in Hodgkin Lymphoma?

Abstract The Epstein-Barr virus (EBV) is a gamma-herpesvirus that colonizes the B-cell system of its human host, allowing it to persist asymptomatically in the majority of the world’s adult population. In most people primary infection goes unnoticed, whereas in a minority of individuals, primary infection results in infectious mononucleosis (IM), a benign condition that almost always resolves after several weeks or months. However, EBV is also causally linked with a number of malignancies, including B-cell lymphomas, such as classical Hodgkin lymphoma (cHL). A proportion of patients with cHL harbor EBV within their tumor cells. Emerging evidence suggests that while EBV is able to subvert cellular processes to promote the growth and survival of HRS cells or their progenitors, mutations in key cell signalization pathways are probably required to do this when EBV is absent. The challenge is to unravel exactly how EBV and its latent genes contribute to the pathogenesis of cHL particularly with respect to how the virus co-operates with cellular genetic and epigenetic changes to drive transformation. It is hoped that the development of better in vitro and in vivo models of disease will reveal more fundamental aspects of EBV’s role in Hodgkin lymphoma pathogenesis and pave the way for targeted therapies for patients with EBV-positive cHL.

Targeting Cancer Cells and Tumor Microenvironment in Preclinical and Clinical Models of Hodgkin Lymphoma Using the Dual PI3Kδ/γ Inhibitor RP6530.

Tumor-associated macrophages (TAMs) and the hyperactivation of the PI3K/AKT pathway are involved in the pathogenesis of Hodgkin lymphoma and affect disease outcome. Because the δ and γ isoforms of PI3K are overexpressed in Hodgkin/Reed-Sternberg (HRS) cells and the tumor microenvironment (TME), we propose that the PI3Kδ/γ inhibitor RP6530 might affect both HRS cells and TME, ultimately leading to an enhanced antitumor response. Hodgkin lymphoma cell lines (L-540, KM-H2, and L-428) and primary human macrophages were used to investigate the activity of RP6530 in vitro and in vivo in Hodgkin lymphoma cell line xenografts. In vitro, RP6530 besides killing and inhibiting the proliferation of Hodgkin lymphoma cells, downregulated lactic acid metabolism, switching the activation of macrophages from an immunosuppressive M2-like phenotype to a more inflammatory M1-like state. By RNA sequencing, we define tumor glycolysis as a specific PI3Kδ/γ-dependent pathway implicated in the metabolic reprogramming of cancer cells. We identify the metabolic regulator pyruvate kinase M2 as the main mediator of tumor-induced immunosuppressive phenotype of macrophages. Furthermore, we show in human tumor xenografts that RP6530 repolarizes TAMs into proinflammatory macrophages and inhibits tumor vasculature, leading to tumor regression. Interestingly, patients with Hodgkin lymphoma experiencing objective responses (complete response and partial response) in a phase I trial using RP6530 showed a significant inhibition of circulating myeloid-derived suppressor cells and an average mean reduction in serum thymus and activation-regulated chemokine levels of 40% (range, 4%-76%). Our results support PI3Kδ/γ inhibition as a novel therapeutic strategy that targets both malignant cells and the TME to treat patients with Hodgkin lymphoma.

Hodgkin lymphoma

Hodgkin lymphoma is characterized by CD30 overexpression and downstream NF-κB pathway activation in Hodgkin/Reed-Sternberg (H/RS) cells. Accordingly, CD30 and its aberrant downstream signaling have been investigated as potential treatment targets for the disease. Given that H/RS cells are surrounded by a variety of immune cells that constitute a unique inflammatory and immunoinhibitory microenvironment, efforts have also been made to treat the disease by reversing the abnormal tumor immune milieu. In recent years, a CD30 antibody-drug conjugate and immune checkpoint inhibitors have been approved for the treatment of Hodgkin lymphoma and have demonstrated remarkable treatment efficacy. These new agents have not only contributed to improved survival of patients refractory to conventional chemotherapy but have also enabled further understanding on the molecular pathogenesis of Hodgkin lymphoma in relation to the key signaling of the PD-1 and JAK/STAT pathways.

Unraveling the Landscape of Copy Number Aberrations in Hodgkin Lymphoma: A Joint KU Leuven and Lysa Study on Circulating Cell Free DNA

Introduction. For decades, the study of genomic alterations in Hodgkin lymphoma (HL) has been hampered by the low abundance of the malignant Hodgkin Reed-Sternberg (HRS) cells in HL lymph node biopsies. Laser microdissection or flow cytometric cell sorting allow the study of purified HRS cells, but the use of these applications is restricted to specialized research centers. We have recently demonstrated proof-of-principle that copy number aberrations (CNA) in HRS cells can be retrieved in circulating cell-free DNA (cfDNA) of HL patients (Vandenberghe et al., Lancet Haematol. 2015). Taking advantage of the presence of HRS cell-derived DNA (ctDNA) in plasma, the aim of this study was to catalogue CNA in HL in a large series of prospectively recruited HL patients.Methods. We analyzed plasma collected from 177 patients (median age 29, range 3-86) with newly diagnosed HL. 60 cases were diagnosed at our institution, including all disease subtypes and stages, with a majority of nodular sclerosis. 118 patients were recruited in the context of the BREACH study, a multicentric Phase 2B study for unfavorable early classical HL (NCT02292979). After cfDNA extraction, samples were low-pass sequenced (0.1x coverage) and analyzed using ichorCNA, an algorithm that produces read depth-based log2 CNA profiles and quantifies the cfDNA tumor content.Results. At diagnosis, 86 % (152/177) of patients showed obvious genomic imbalances in cfDNA, in early-stage (85 % (131/155)) as well as in advanced cases (95 % (21/22)). Among the abnormal profiles, gains encompassing 2p16, 5p15, 9p24, 12q13 and 19q13 were the most frequent aberrations, occurring in 79 %, 53 %, 57 %, 63 % and 80 % respectively. Losses most frequently affected regions 4q34, 6q23, 11q22 and 13q13, in 53 %, 63 %, 49 % and 59 % of abnormal profiles respectively (fig. 1A). All these CNAs have been previously described with varying frequencies in smaller series of 10-53 patients, using arrayCGH or whole exome sequencing on microdissected HRS cells or even HRS cell-derived cell lines as input material. The observed pattern of CNAs is distinct from the pattern we observed in cfDNA obtained from other hematological malignances, e.g. multiple myeloma and diffuse large B-cell lymphoma (fig. 1B, C).Analysis of follow-up cfDNA samples revealed that the majority of patients rapidly clear their profiles during the first two treatment cycles. Of 123 samples analysed at d15 of cycle 1, 89 samples (72 %) showed a normal profile without CNAs.This suggests a relationship between the fraction of ctDNA in cfDNA and the disease burden. However, we could not find a correlation between the ctDNA fraction as calculated by ichorCNA and the metabolic tumor volume as computed from PET/CT scans. This could be due to several factors, e.g. the small fraction of HRS cells in the metabolic tumor volume. We are currently investigating whether a correlation does exist between the HRS content in the lymph node biopsy and the ctDNA load.Conclusions. In this largest study of CNA in HL to date, we provide a comprehensive catalogue of the types of CNAs, as well as their frequencies and patterns in HL. In this series of 177 patients, gains affecting 2p, 12q and 19q and loss of 6q and 13q emerge as the most commonly recurrent CNAs in HL, across all HL subtypes and stages. The data confirm and extend our previous findings that the majority of HL patients, including those with early-stage disease, have detectable CNAs in their cfDNA at diagnosis. We demonstrate a broad spectrum of aberrations, gains and losses, some of which recur at higher frequencies than gains of 9p24, harboring PD-L1. This warrants further studies on how these CNAs are implicated in the pathogenesis of HL. It further endorses the use of ctDNA as an alternative gateway to the genome of HRS cells, and as a substrate for the evaluation of early disease response. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.