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Primary mediastinal B-cell lymphoma (PMBCL) is a rare but aggressive mature B-cell lymphoma that arises from thymic B cells and most commonly affects adolescents and young adults. PMBCL is now recognized by the WHO as a distinct entity from diffuse large B-cell lymphoma (DLBCL), not otherwise specified, with a unique clinical presentation and distinct morphologic features and molecular alterations. Similar to classic Hodgkin lymphoma, PMBCL tumors are characterized by alterations in the nuclear factor-κB and JAK/STAT pathways. These tumors also exhibit an immune evasion phenotype marked by upregulation of PD-L1 and loss of B2M. Historic data indicates that outcomes for pediatric patients with PMBCL are inferior compared with pediatric patients with DLBCL treated on the same protocols, and there is no current standard approach to initial treatment. Common regimens used for children with PMBCL include multiagent chemotherapy regimens designed for Burkitt lymphoma, such as Lymphomes Malins B (LMB)-based or Berlin-Frankfurt-Münster (BFM)-based chemotherapy ± rituximab. Based on initial data in adults showing excellent outcomes with the use of DA-EPOCH-R regimens, these regimens have also been adopted in pediatrics, although with mixed results. Novel agents are currently being studied in PMBCL with the goal of improving outcomes and reducing reliance on radiation and/or high-dose chemotherapy. Immune checkpoint blockade with PD-1 inhibition is of particular interest given the upregulation of PD-L1 in PMBCL and the known efficacy of these agents in the relapsed setting. Future efforts in PMBCL will also seek to determine the role of FDG-PET in evaluating response to therapy and the role of biomarkers in risk stratification.

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis. Significance: Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains. This article is highlighted in the In This Issue feature, p. 171

R code of our analysis clock like mutational signatures in Hodgkin lymphoma WGS and WES.

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis.Significance:Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains.This article is highlighted in the In This Issue feature, p. 171

All supplementary figures S1-S17

R code of our analysis for driver mutation discovery in Hodgkin lymphoma WGS and WES.

Supplementary Tables S1-S20

Supplemental Figure Legends

PU-H71 suppresses tumor growth in a Ramos xenograft Model.

IPA analysis of Hps90 client protein networks

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity in vitro and in vivo, including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition in vitro and in vivo. Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. Mol Cancer Ther; 16(9); 1779–90. ©2017 AACR.

Hsp90 client proteins identified by PU-H71 high affinity capture

BL tumors undergo growth suppression without apoptosis upon treatment with PU-H71 in-vivo.

BL tumors overexpress Hsp90

BL cells but not normal GC B-cells are sensitive to inhibition by PU-H71.

Chemical Structure of PU-H71 and BEZ-235

Outcomes for children and adolescents with relapsed and refractory Hodgkin lymphoma (HL) are poor, with approximately 50% of patients experiencing a subsequent relapse. The anti-CD30 antibody-drug conjugate brentuximab vedotin improved progression-free survival (PFS) when used as consolidation after autologous stem cell transplant (ASCT) in adults with high-risk relapsed/refractory HL. Data on brentuximab vedotin as consolidative therapy after ASCT in pediatric patients with HL are extremely limited, with only 11 patients reported in the literature. We performed a retrospective analysis of 67 pediatric patients who received brentuximab vedotin as consolidation therapy following ASCT for the treatment of relapsed/refractory HL to describe the experience of this regimen in the pediatric population. This is the largest cohort reported to date. We found that brentuximab vedotin was well tolerated with a safety profile similar to adult patients. With a median follow up of 37 months, the 3-year PFS was 85%. These data suggest a potential role for the use of brentuximab vedotin as consolidation therapy after ASCT for children with relapsed/refractory Hodgkin lymphoma.

Despite advances in T-cell immunotherapy against Epstein-Barr virus (EBV)-infected lymphomas that express the full EBV latency III program, a critical barrier has been that most EBV+ lymphomas express the latency I program, in which the single Epstein-Barr nuclear antigen (EBNA1) is produced. EBNA1 is poorly immunogenic, enabling tumors to evade immune responses. Using a high-throughput screen, we identified decitabine as a potent inducer of immunogenic EBV antigens, including LMP1, EBNA2, and EBNA3C. Induction occurs at low doses and persists after removal of decitabine. Decitabine treatment of latency I EBV+ Burkitt lymphoma (BL) sensitized cells to lysis by EBV-specific cytotoxic T cells (EBV-CTLs). In latency I BL xenografts, decitabine followed by EBV-CTLs results in T-cell homing to tumors and inhibition of tumor growth. Collectively, these results identify key epigenetic factors required for latency restriction and highlight a novel therapeutic approach to sensitize EBV+ lymphomas to immunotherapy.

SummaryEpstein-Barr virus (EBV) subverts host epigenetic pathways to switch between viral latency programs, colonize the B-cell compartment and reactivate. Within memory B-cells, the reservoir for lifelong infection, EBV genomic DNA and histone methylation marks restrict gene expression. But, this epigenetic strategy also enables EBV-infected tumors, including Burkitt lymphomas to evade immune detection. Little is known about host cell metabolic pathways that support EBV epigenome landscapes. We therefore used amino acid restriction, metabolomic and CRISPR approaches to identify that an abundant methionine supply, and interconnecting methionine and folate cycles, maintain Burkitt EBV gene silencing. Methionine restriction, or methionine cycle perturbation, hypomethylated EBV genomes, de-repressed latent membrane protein and lytic gene expression. Methionine metabolism also shaped EBV latency gene regulation required for B-cell immortalization. Dietary methionine restriction altered murine Burkitt xenograft metabolomes and de-repressed EBV immunogens in vivo. These results highlight epigenetic/immunometabolism crosstalk supporting the EBV B-cell lifecycle and suggest therapeutic approaches.HighlightsMethionine metabolism is critical for Epstein-Barr virus B-cell latencyExtensive cross-talk enables methionine metabolism to control the EBV epigenomeMethionine restriction also impairs EBV-driven human B-cell immortalizationDietary methionine restriction unmasks EBV antigens in Burkitt xenografts in vivo

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells within a classic Hodgkin lymphoma (cHL) biopsy limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Here we leveraged this method to report the first whole genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events prior to the clinical diagnosis of cHL. We identified alterations in driver genes not previously described in cHL, a high activity of the APOBEC mutational signature, and the presence complex structural variants including chromothripsis. We found that the high ploidy observed in cHL is often acquired through multiple, independent large chromosomal gain events including whole genome duplication. The first of these likely occurs several years prior to the diagnosis of cHL, and the last gains typically occur very close to the time of diagnosis. Evolutionary timing analyses revealed that driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID driven mutagenesis usually preceded large chromosomal gains. The study provides the first temporal reconstruction of cHL pathogenesis and suggests a relatively long time course between the first pathogenic event and the clinical diagnosis.

Lymphoma is the one of the most common cancer diagnoses among adolescents and young adults (AYAs) aged 15–39. Despite significant advances in outcomes observed in older adults and younger children, improvements in AYAs have lagged behind. The reasons for this are likely multifactorial including disparities in access to health insurance, low rates of enrollment to clinical trials, potential differences in disease biology, and unique psychosocial challenges. Here we will review Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL), two of the most common aggressive lymphomas that occur in AYAs. We will discuss the current knowledge about disease biology in AYAs, adult and pediatric treatment strategies, novel targeted therapies, and ongoing AYA clinical trials in these lymphoma subtypes. We also will review unique considerations for treatment-related toxicities in AYAs and psychosocial issues relevant to this population. Pediatric and adult trials in HL and PMBCL have demonstrated that treatment with dose-intense chemotherapeutic regimens with or without radiation results in high cure rates but can also be associated with long-term toxicity which must be considered in this young population. Novel targeted agents such as the antibody-drug conjugate brentuximab vedotin and/or antibodies targeted against PD-1/PD-L1 have demonstrated activity in the relapsed setting and are currently being evaluated in the upfront setting, which may reduce our reliance on therapies associated with long-term toxicity. AYA-focused clinical trials are currently underway to better elucidate the optimal therapy for lymphomas in this age group. There is an urgent need for clinical trials including AYAs in order to increase the knowledge of age-specific outcomes, toxicities, disease biology, and the need to develop comprehensive AYA care models that meet the unique and complex care needs of this patient population.

Molecular alterations in the histone methyltransferase EZH2 and the antiapoptotic protein Bcl-2 frequently co-occur in diffuse large B-cell lymphoma (DLBCL). Because DLBCL tumors with these characteristics are likely dependent on both oncogenes, dual targeting of EZH2 and Bcl-2 is a rational therapeutic approach. We hypothesized that EZH2 and Bcl-2 inhibition would be synergistic in DLBCL. To test this, we evaluated the EZH2 inhibitor tazemetostat and the Bcl-2 inhibitor venetoclax in DLBCL cells, 3-dimensional lymphoma organoids, and patient-derived xenografts (PDXs). We found that tazemetostat and venetoclax are synergistic in DLBCL cells and 3-dimensional lymphoma organoids that harbor an EZH2 mutation and an IGH/BCL2 translocation but not in wild-type cells. Tazemetostat treatment results in upregulation of proapoptotic Bcl-2 family members and priming of mitochondria to BH3-mediated apoptosis, which may sensitize cells to venetoclax. The combination of tazemetostat and venetoclax was also synergistic in vivo. In DLBCL PDXs, short-course combination therapy resulted in complete remissions that were durable over time and associated with superior overall survival compared with either drug alone.

Survivors of Hodgkin lymphoma (HL) have an increased risk of subsequent malignant neoplasms (SMNs). Response-adapted treatment may decrease this risk by reducing exposure to therapy associated with SMN risk. The Children’s Oncology Group study AHOD0031 evaluated response-adapted therapy for children and adolescents with intermediate-risk HL. We report the SMNs among 1711 patients enrolled in AHOD0031. Patients were treated with 4 cycles of doxorubicin, bleomycin, vincristine, etoposide, prednisone, and cyclophosphamide with or without involved-field radiation therapy (RT). Patients with a slow early response to initial chemotherapy were randomized to 2 additional cycles of dexamethasone, etoposide, cisplatin and cytarabine or no additional chemotherapy, and all received RT. At a median follow-up of 7.3 years, an analysis of SMNs was performed. The 10-year cumulative incidence of SMN was 1.3% (95% confidence interval [CI], 0.6-2.0). SMNs included 3 patients with acute myeloid leukemia (AML), 11 with solid tumors, and 3 with non-Hodgkin lymphoma. Sixteen of 17 patients with an SMN had received combined modality therapy. The standardized incidence ratio for SMN was 9.5 (95% CI, 4.5-15.2) with an excess absolute risk of 1.2 per 1000 person-years. The cumulative incidence of SMNs was higher among patients who received RT (P = .037). In multivariate analysis, RT, B symptoms, and race were associated with SMN risk. Given the latency from exposure, we have likely captured all cases of secondary leukemia and myelodysplastic syndrome (MDS). Longer follow-up is needed to determine the risk of solid tumors. Avoidance of RT without sacrificing disease control should remain a goal for future therapeutic approaches. This trial was registered at www.clinicaltrials.gov as #NCT00025259.

Epstein-Barr virus (EBV) is associated with multiple human malignancies. To evade immune detection, EBV switches between latent and lytic programs. How viral latency is maintained in tumors or in memory B-cells, the reservoir for lifelong EBV infection, remains incompletely understood. To gain insights, we performed a human genome-wide CRISPR/Cas9 screen in Burkitt lymphoma B-cells. Our analyses identified a network of host factors that repress lytic reactivation, centered on the transcription factor MYC and including cohesins, FACT, STAGA and Mediator. Depletion of MYC or factors important for MYC expression reactivated the lytic cycle, including in Burkitt xenografts. MYC bound the EBV genome origin of lytic replication and suppressed its looping to the lytic cycle initiator BZLF1 promoter. Notably, MYC abundance decreases with plasma cell differentiation, a key lytic reactivation trigger. Our results suggest that EBV senses MYC abundance as a readout of B-cell state and highlights Burkitt latency reversal therapeutic targets.

Dysregulation of the histone methyltransferase EZH2 plays a critical role in the development of a variety of malignancies including B-cell lymphomas. As a result, a series of small molecule inhibitors of EZH2 have been developed and studied in the pre-clinical setting. Three EZH2 inhibitors: tazemetostat (EPZ-6438), GSK2816126 and CPI-1205 have moved into phase I/phase II clinical trials in patients with non-Hodgkin lymphoma and genetically defined solid tumors. Early data from the tazemetostat trials indicate an acceptable safety profile and early signs of activity in diffuse large B-cell lymphoma and follicular lymphoma, including patients with EZH2 wild-type and mutant tumors. In this review, we present the rationale, key pre-clinical and early clinical findings of small molecule EZH2 inhibitors for use in lymphoma as well as future challenges and potential opportunities for combination therapies.

Background: Although the prognosis for children with Hodgkin lymphoma (HL) is excellent, up to 25% of patients experience relapse and require salvage therapy. Event-free survival (EFS) for patients with relapsed or refractory disease (R/R) who require high-dose therapy with autologous stem cell transplantation (ASCT) is between 31-67% (Daw, 2011), and efforts to improve these outcomes are an area of urgent clinical need. Brentuximab vedotin (BV) is an anti-CD30 antibody-drug conjugate combined with monomethyl auristatin E. The phase III AETHERA study (NCT01100502) was a randomized, placebo-controlled trial that demonstrated consolidation with BV following ASCT for adults with R/R HL improved progression-free survival (PFS) in comparison to placebo (5-year PFS 59% vs. 41%) in patients identified as having a high-risk of post-ASCT progression (Moskowitz, 2015, 2018). However, there is limited data on post-ASCT BV consolidation in pediatric patients. Here, we report a retrospective multi-center study of BV as consolidation post-ASCT in pediatric patients with R/R HL. Patients and Methods: We performed a retrospective analysis of pediatric patients aged ≤ 21 years who received BV as consolidative therapy following ASCT for the treatment of relapsed/refractory HL. Data was collected on disease risk factors (refractory disease or relapse ≤ 12 months after frontline therapy, best response of partial remission or stable disease to salvage therapy, presence of extranodal involvement, presence of B symptoms, and number of systemic treatments pre-ASCT ≥ 2), treatment history, BV-related toxicity, and response to therapy. The primary endpoint was 3-year EFS. Events were defined as relapse, progression of disease, or death from any cause. Results: Seventy patients were identified from 14 academic centers. Eighteen received BV >90 days after ASCT and were excluded due to significant delay in initiation of BV. Of the remaining 52 patients, the median age at diagnosis was 15 years (range 4-21), and the median age at diagnosis of R/R disease was 16 years (range 8-22). Twenty-eight patients (54%) were male, 13 (25%) had B symptoms, 19 (37%) had extranodal disease. Six patients (12%) had primary refractory disease and 24 patients (47%) relapsed The median time from ASCT to the start of BV consolidation was 52 days (range 30-90 days). The most frequent adverse events were peripheral neuropathy of any grade (sensory 19/52 [37%]; motor (14/52 [27%]), and cytopenias (grade 3 or 4: (25/52 [48%]), including neutropenia (13/52 [25%]), thrombocytopenia (7/52 [14%]), and anemia (5/52 [10%]). Three patients (6%) experienced infusion-related reactions. The median number of BV cycles completed post-ASCT was 12 (range 1-17), and 16 patients (31%) completed the full 16 cycles of BV. The most common reason for premature termination was adverse effects (22/36 [61%]). Additional reasons included: physician plan for shorter duration (4/36 [11%]), patient/family decision (3/36 [8%]), and progression (1/36 [3%]). Treatment was still ongoing in 3 patients and reason for premature termination was not reported for 17/36 patients (47%). With a median follow of 2.8 years (range 0.1 to 6.25 years) the three-year EFS was 92% [95%CI: 83-100]. Patients with 0-1 risk factors (15/52 [29%]) and ≥ 2 risk factors (37/52 [71%]), had a 3-year EFS of 100% and 90% [95%CI: 79-100], respectively. Fifty patients (96%) are alive with no evidence of disease, while 2 (4%) patients are alive with disease. No deaths have occurred. Conclusion: The use of post-ASCT BV consolidation for pediatric patients with R/R HL is associated with a favorable safety and tolerability profile. In this cohort, the combination of salvage therapy, HD-chemotherapy/ASCT, and BV consolidation resulted in extremely promising outcomes and warrants further investigation in a prospective pediatric trial. Figure 1 Figure 1. Disclosures Leger: Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbott Diagnostics: Research Funding. Roth: Merck: Consultancy; Janssen: Consultancy.

While Epstein-Barr virus (EBV) and the Kaposi sarcoma herpesvirus (KSHV)/human herpesvirus (HHV) 8 have shown a definite association with lymphoproliferative disease, a role for the HHV-6 has been less clear. Similar to other herpesviruses, HHV-6 predominantly remains latent following initial infection, but can be reactivated during stress or immune suppression, and is the cause of roseola in young children. Existing as two distinct species, HHV-6B is more common, infecting ~90% of adults. HHV-6B, a T-lymphotropic virus, enters cells via CD134, a TNF receptor superfamily member, expressed on both naïve and CD4 +CD25 + T cells, leading to CD4 + lymphocyte depletion and impaired T cell activation. HHV-6 has been variably detected in classic Hodgkin (CHL) and T-cell lymphomas (TCL) by immunohistochemistry (IHC) and PCR with more recent data suggesting infection may be confined to tumor-associated lymphocytes. The specificity of these IHC antibodies is not well documented. The question remains whether HHV-6 in the tumor microenvironment of advanced disease is a consequence of immune dysfunction, or may play a more direct role in tumor initiation and progression by altering the tumor microenvironment. To address these questions, we evaluated HHV-6B viral gene expression patterns in lymphoma patient samples by RNA sequencing techniques. Following IRB approval, CHL, TCL, B-cell, and post-transplant lymphoproliferative disease (PTLD) cases were screened for potential HHV-6-association by IHC with an antibody against HHV-6 gp60/110 envelope glycoprotein (Millipore Sigma, MAB8537). Positive cases with available frozen tissue and adequate RNA (5) or sorted T-cell subsets from Hodgkin lymphoma (11) underwent bulk RNA-seq (rRNA depletion (Illumina), 50M reads/sample). Viral transcripts were identified by performing the Burrows-Wheeler Alignment by reference host alignment (to filter host and bad quality reads) followed by viral reference host alignment. Previous TCL databases with available RNAseq data were similarly evaluated. IHC analysis revealed 5/25 CHL, 34/52 TCL, 5/13 PTLD, 4/81 diffuse large B-cell lymphoma (DLBCL) and 2/28 follicular lymphoma (FL) with rare gp60/110-positive cells. This included 11 CHL cases with sorted T-cell subsets, of which one showed membranous and Golgi gp60/110 staining in background T-cells (25-year-old female, nodular sclerosis subtype, EBV-negative). Of these 11 CHL cases, RNAseq of T-cell subsets revealed a pattern of HHV-6B transcripts in only this case. Frozen tumor blocks were available from 5 additional cases with positive gp60/110 staining (2 CHL, 1 DLBCL, 1 FL and 1 PTLD), but RNAseq analysis did not identify any HHV-6B transcripts. Notably, these cases had dim cytoplasmic but not Golgi gp60/110 staining. RNA sequencing data derived from two independent TCL cohorts were analyzed for HHV-6B transcripts. Although no HHV-6B transcripts were detected via RNAseq in 20 angioimmunoblastic T-cell lymphoma samples from one TCL cohort, many had EBV-gene expression. HHV-6B transcripts were detected in two cases of anaplastic large cell lymphoma (ALCL) in a second TCL cohort (2/79 cases). High expression of the U67, U68, U79 and U90 genes was found, revealing overlap of the HHV-6B transcript expression between ALCL and CHL samples (Fig 1). Additionally, detection of two genes that could be driving tumor growth (U51, which encodes a G-protein receptor and U24, which inhibits proper T cell activation, reducing secretion of cytokines at infection site) demonstrates a specific viral gene expression pattern within the intratumor T-cell population. The potential presence of HHV-6B infection in the lymphoma microenvironment is controversial. To our knowledge, this is the first report conclusively demonstrating HHV-6B expression in CHL using RNAseq. Notably, the viral gene expression pattern seen in CHL overlaps with that found in two cases of ALCL, highlighting viral proteins of potential particular significance. These data may aid in development of a more reliable means of HHV-6B detection. For example, the immediate early gene U90, a transcriptional activator that may induce expression of both viral and cellular genes that affect the tumor microenvironment, was consistently expressed and may be a reliable marker of HHV-6B infection. Funding: HHV-6 Foundation Figure 1 Figure 1. Disclosures Tychinin: BostonGene Inc.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Karelin: BostonGene Inc.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Cherdintsev: BostonGene Inc.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Kudryashova: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Egorov: BostonGene: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Degryse: BostonGene Inc.: Current Employment, Current holder of stock options in a privately-held company. Kotlov: BostonGene Corp: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties. Bagaev: BostonGene Corp.: Current Employment, Current holder of stock options in a privately-held company, Patents & Royalties: BostonGene. Roth: Merck: Consultancy; Janssen: Consultancy. Roshal: Celgene: Other: Provision of services; Physicians' Education Resource: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services. Rabadan: Genotwin: Other: Raul Rabadan is founder of Genotwin; AimedBio: Membership on an entity's Board of Directors or advisory committees. Elemento: Owkin: Consultancy, Other: Current equity holder; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Janssen: Research Funding; Eli Lilly: Research Funding; Champions Oncology: Consultancy; AstraZeneca: Research Funding; Johnson and Johnson: Research Funding.

Introduction: Immunotherapy is a promising therapeutic intervention for cancer treatment. Activation of the immune system via checkpoint blockade has been shown to produce antitumor responses in patients with both solid and hematological tumors. However, many patients do not respond to checkpoint inhibitors, and additional therapies are needed to treat these patients. Testing immunotherapies requires a functional human immune system; thus, it is difficult to evaluate their effectiveness using conventional experimental models. For this reason, establishing in vivo models that closely reproduce not only human tumors, but also their interactions with the human immune system, has become mandatory. Methods: We developed a humanized mouse model and combined it with a patient-derived tumor xenograft (PDTX). Humanized mice (HuMice) were generated by transplantation of cord blood or mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells into preconditioned immunodeficient mice. We compared human engraftment in 3 different mouse strains: NSG (NOD.Cg-Prkdc scidIl2rg tm1Wjl/SzJ), NSGS (NOD.Cg-Prkdc scidIl2rg tm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ) and NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl/ThomJ). Immune cell profiling and distribution was performed using flow cytometry and immunohistochemistry. The B cell receptor (BCR) repertoire was evaluated using an RNA-based NGS assay. To evaluate the maturation and functionality of T cells developing in HuMice we performed proliferation, degranulation and intracellular cytokine staining. Results: Two months after CD34+ cell transplantation, we observed high levels of human hematopoietic chimerism in all the 3 strains. NSGS mice supported high-level chimerism as early as 1 month after transplantation, with more than 25% of human CD45+ cells in the blood. In all mice the majority of human circulating leukocytes were CD19+ B cells. An early appearance of CD3+ human T cells was detected in NSGS mice as compared to the other strains. Notably, the T cell expansion correlated with a decrease in relative B cell abundance while the myeloid cell contribution to the graft remained steady. We documented the differentiation of CD4+ and CD8+ human T cells at a 2:1 ratio. The characterization of the T cell subsets revealed that the majority was represented by CD45RA-CCR7- effector memory cells in both the spleen and the blood of HuMice. Nevertheless, recipient mice did not exhibit overt signs of graft-versus-host disease. We also evaluated the cytotoxic potential of T cells isolated from the spleen of HuMice: ex vivo peptide antigen (i.e. EBV) presentation let to generation of effective and specific cytotoxic T-cells. After assessing a functional human immune system reconstitution in HuMice, we challenged them in vivo with low-passage tumor fragments from a diffuse large B cell lymphoma (DLBCL) PDTX. All tumor implants were successfully engrafted in both HuMice and non-humanized controls. Remarkably, all the 3 HuMice strains showed a significant reduction in the tumor volume and/or eradication compared to matched non-humanized controls. Flow cytometry analysis of the peripheral blood of humanized PDTX revealed that the tumor engraftment elicited a significant expansion of CD3+ T cells and cytotoxic CD8+ lymphocytes. Moreover, tumors developing in HuMice exhibited intermediate to high levels of tumor infiltrating T lymphocytes commingling with the neoplastic B cells, as determined by immunohistochemistry. Large areas of necrosis were often observed in PDTX of HuMice. Infiltrating CD3+ cells were TIGIT, PD-1 and Lag-3 positive, and did not efficiently proliferate ex vivo: all features consistent with an exhaustion phenotype. PDTX of HuMice often displayed larger areas of necrosis. Conclusions: Collectively, our data demonstrate that a robust reconstitution can be achieved in different strains of immunocompromised mice and that HuMice elicit effective anti-lymphoma responses. PDTX HuMice represent a powerful platform to study host-tumor interactions, and to test novel immune-based strategies (CAR-T, bifunctional Abs) and new pharmacological approaches to counteract T-cell exhaustion. Figure 1 Figure 1. Disclosures Scandura: Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Constellation: Research Funding; MPN-RF (Foundation): Research Funding; CR&T (Foudation): Research Funding; European Leukemia net: Honoraria, Other: travel fees . Roth: Janssen: Consultancy; Merck: Consultancy.

Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a small fraction of Hodgkin and Reed-Sternberg (HRS) tumor cells (~1%) which are surrounded by an extensive immune infiltrate. The rare nature of HRS cells limits the ability to study the genomics of cHL using standard platforms. To circumvent this, our group has optimized fluorescence-activated cell sorting to isolate HRS cells and intratumor B- and T- cells and to perform whole exome sequencing (WES; Reichel, Blood 2015). To date, however, there have been no reports on whole genome sequencing (WGS) of cHL. Methods: We performed flow-sorting of HRS cells and WGS to define the genomic landscape of cHL including: i) mutational processes involved in pathogenesis, ii) large and focal copy number variants, iii) structural variants including complex events, iv) the sequence and evolution of molecular events in cHL. We interrogated WGS from 25 cases of cHL: 10 pediatric patients (age40). Intra-tumoral T-cells were used as germline control. An additional 36 cHL cases were evaluated by WES. Results: The average depth of coverage among the 25 WGS cases was 27.5x. After having identified and removed amplification-based palindromic sequencing artifacts, we observed a median of 5006 single base substitutions (SBS; range 1763-18436). Pediatric and AYA patients had a higher SBS burden compared to older adults (median 5279 vs. 2945, p=0.009). Five main SBS signatures were identified: SBS1 and SBS5 (aging), SBS2 and SBS13 (APOBEC), and SBS25 (chemotherapy, in a relapsed case). A dNdScv driver discovery analysis performed on the combined WES and WGS cases identified 24 driver genes including BCL7A and CISH which had not been previously reported as drivers in cHL. An investigation of copy number alterations (CNAs) confirmed high ploidy in cHL (median 2.95, range 1.66-5.33). Whole genome duplication was identified in 64% cases. We also observed clear evidence of complex events such as chromothripsis (n=4), double minutes (dm, n=2), breakage-fusion-bridge (bfb; n=4). Some of these events were responsible for the acquisition of distinct drivers. For example, we observed one dm and one bfb responsible for CD274 and REL gains, respectively (>10 copies). Leveraging the high prevalence of large chromosomal gains, we performed an investigation of the relative timing of acquisition of driver mutations. Clonal mutations within chromosomal gains can be defined as duplicated (VAF~66%; acquired before the gain) or non-duplicated (VAF~33%; acquired before or after the gain). Sixty-one percent (152/249) of driver genes were duplicated suggesting that they were acquired prior to large chromosomal gains. Next, we used the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains to estimate the molecular time of each duplicated segment (Rustad, Nat Comm 2020). In 11/22 genomes the final CNA profile was acquired through at least two temporally distinct events. To convert these relative estimations into absolute timing (i.e., the age at which events occurred), we leveraged the clock-like mutation signatures (SBS1, SBS5). We first confirmed that the SBS1 and SBS5 mutation rate were constant over time (R 2=0.84; p Conclusion: Here we report the first WGS in cHL. We identify novel drivers and genomic mechanisms involved in cHL pathogenesis. We found that mutations in driver genes are often acquired earlier then chromosomal gains, potentially preceding the cHL diagnosis by several years. In addition, we observed key differences in biology of cHL across age groups including accelerated mutagenesis and increased mutational burden among younger patients. Disclosures Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Oberley: Caris LIfe Science: Current Employment. Lim: EUSA Pharma: Honoraria. Landgren: Janssen: Other: IDMC; Celgene: Research Funding; Janssen: Honoraria; Amgen: Honoraria; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Moskowitz: Merck & Co., Inc.: Research Funding. Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Elemento: Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; Champions Oncology: Consultancy; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Janssen: Research Funding. Roth: Janssen: Consultancy; Merck: Consultancy.

Bimiralisib is an orally bioavailable pan-phosphatidylinositol 3-kinase and mammalian target of rapamycin inhibitor which has shown activity against lymphoma in preclinical models. This phase I/II study evaluated the response rate to bimiralisib at 2 continuous dose levels (60 mg and 80 mg) in patients with relapsed/refractory lymphoma. Fifty patients were enrolled and started treatment. The most common histologies were diffuse large B-cell lymphoma (n = 17), follicular lymphoma (n = 9), T-cell lymphoma (n = 8), and others (mostly indolent). Patients had been treated with a median of 5 prior lines of treatment and 44% were considered refractory to their last treatment. Mean duration of treatment (and standard deviations) with 60 mg once daily (o.d.) was 1.3 ± 1.2 months, and with 80 mg o.d. 3.7 ± 3.9 months. On an intention to treat analysis, the overall response rate was 14% with 10% achieving a partial response and 4% a complete response. Thirty-six percent of patients were reported as having stable disease. No dose-limiting toxicities were observed during the phase I portion of the study. Overall, 70% of patients had a grade 3 treatment emergent adverse events (TEAE) and 34% had a grade 4 TEAE; 28% of patients discontinued treatment due to toxicity. The most frequent TEAEs grade ≥3 was hyperglycemia (24%), neutropenia (20%), thrombocytopenia (22%), and diarrhea (12%). Per protocol, hyperglycemia required treatment with oral antihyperglycemic agents in 28% and with insulin in 14%. At 60 mg or 80 mg continuous dosing, bimiralisib showed modest efficacy with significant toxicity in heavily pretreated patients with various histological subtypes of lymphoma.

Summary Treatment with dose-adjusted EPOCH (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone) chemotherapy and rituximab (DA-EPOCH-R) has become the standard of care for primary mediastinal B-cell lymphoma (PMBCL) at many institutions despite limited data in the multi-centre setting. We report a large, multi-centre retrospective analysis of children and adults with PMBCL treated with DA-EPOCH-R to characterize outcomes and evaluate prognostic factors. We assessed 156 patients with PMBCL treated with DA-EPOCH-R across 24 academic centres, including 38 children and 118 adults. All patients received at least one cycle of DA-EPOCH-R. Radiation therapy was administered in 14·9% of patients. With median follow-up of 22·6 months, the estimated 3-year event-free survival (EFS) was 85·9% [95% confidence interval (CI) 80·3–91·5] and overall survival was 95·4% (95% CI 91·8–99·0). Outcomes were not statistically different between paediatric and adult patients. Thrombotic complications were reported in 28·2% of patients and were more common in paediatric patients (45·9% vs. 22·9%, P = 0·011). Seventy-five per cent of patients had a negative fluorodeoxyglucose positron emission tomography (FDG-PET) scan at the completion of DA-EPOCH-R, defined as Deauville score 1–3. Negative FDG-PET at end-of-therapy was associated with improved EFS (95·4% vs. 54·9%, P

Primary central nervous system lymphoma (PCNSL) is a rare subtype of non-Hodgkin lymphoma (NHL) in childhood and adolescence, with an increased risk among patients with immunodeficiency.1-5 Given the rarity and lack of any prospective trials, reliable data on PCNSL are lacking, with most data coming from case reports or small series. Thus, 2 of the largest childhood NHL consortia, the European Intergroup for Childhood Non-Hodgkin Lymphoma (EICNHL) and the international Berlin-Frankfurt-Munster (i-BFM) Group, as well as selected North American centers, designed a retrospective multinational study on pediatric PCNSL. Here, we present data from the largest series of pediatric patients with PCNSL reported to date.

To accomplish the remarkable task of lifelong infection, the Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programmes to navigate the B-cell compartment and evade immune responses. The transforming programme, consisting of highly immunogenic EBV nuclear antigen (EBNA) and latent membrane proteins (LMPs), is expressed in newly infected B lymphocytes and in post-transplant lymphomas. On memory cell differentiation and in most EBV-associated Burkitt's lymphomas, all but one viral antigen are repressed for immunoevasion. To gain insights into the epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR-Cas9 screen was performed in EBV and Burkitt's lymphoma cells. Here, we show that the ubiquitin ligase ubiquitin-like PHD and RING finger domain-containing protein 1 (UHRF1) and its DNA methyltransferase partner DNA methyltransferase I (DNMT1) are critical for the restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency programme switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal centre B cells, the Burkitt's lymphoma cell of origin, providing a molecular link between B-cell state and the EBV latency programme. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.

Purpose: Enhancer of zeste homolog 2 (EZH2) activity is dysregulated in many cancers. Patients and Methods: This phase I study determined the safety, maximum-tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of the intravenously administered, highly selective EZH2 inhibitor, GSK2816126, (NCT02082977). Doses of GSK2816126 ranged from 50 to 3,000 mg twice weekly, and GSK2816126 was given 3-weeks-on/1-week-off in 28-day cycles. Eligible patients had solid tumors or B-cell lymphomas with no available standard treatment regimen. Results: Forty-one patients (21 solid tumors, 20 lymphoma) received treatment. All patients experienced ≥1 adverse event (AE). Fatigue [22 of 41 (53.7%)] and nausea [20 of 41 (48.8%)] were the most common toxicity. Twelve (32%) patients experienced a serious AE. Dose-limiting elevated liver transaminases occurred in 2 of 7 patients receiving 3,000 mg of GSK2816126; 2,400 mg was therefore established as the MTD. Following intravenous administration of 50 to 3,000 mg twice weekly, plasma GSK2816126 levels decreased biexponentially, with a mean terminal elimination half-life of approximately 27 hours. GSK2816126 exposure (maximum observed plasma concentration and area under the plasma-time curve) increased in a dose-proportional manner. No change from baseline in H3K27me3 was seen in peripheral blood mononuclear cells. Fourteen of 41 (34%) patients had radiological best response of stable disease, 1 patient with lymphoma achieved a partial response, 21 of 41 (51%) patients had progressive disease, and 5 patients were unevaluable for antitumor response. Conclusions: The MTD of GSK2816126 was established at 2,400 mg, but the dosing method and relatively short half-life limited effective exposure, and modest anticancer activity was observed at tolerable doses.

Paediatric B-cell non-Hodgkin lymphoma (B-NHL) compromises a heterogeneous group of histological entities of which Burkitt lymphoma is the most common. In resource-rich countries, the expected cure rate is in excess of 85% with application of risk-adapted short intensive chemotherapy. In recent years, large paediatric cooperative group trials have sought to improve upon outcomes by decreasing the intensity of cytotoxic treatment as well as introducing targeted therapies, such as rituximab. These efforts have resulted in excellent outcomes, however there remains a group of high-risk patients for whom novel treatment approaches are needed. In this review, we will summarize the recent paediatric clinical trials in B-NHL as well as compare treatment approaches across the major cooperative groups. We will also highlight our current understanding of the molecular biology of paediatric B-NHL with a focus on how this may help guide future rational targeted therapy.

Hodgkin lymphoma (HL) and primary mediastinal B-cell lymphoma (PMBCL) are both B-cell malignancies that commonly arise in the mediastinum and have a peak incidence in the adolescent and young adult (AYA) age range of 15-39y. Advances in sequencing technology with low-input samples have allowed for recent advances in our knowledge of the key molecular alterations in these lymphoma subtypes. In this session, we will discuss the unique genomic features of HL and PMBCL and highlight what is known about differences across the age spectrum. We will also discuss how genomics can inform the use of novel therapies for HL and PMBCL. Hodgkin lymphoma has traditionally been challenging to study by standard genomic platforms given the rarity of the Hodgkin Reed Sternberg (HRS) cell among the extensive inflammatory infiltrate. Recently, our group and others have utilized flow cytometry to sort HRS cells and intratumoral T and B cells for genomic analyses. Optimization of library preparation for ultralow ( Primary mediastinal B-cell lymphoma is a rare subtype of non-Hodgkin lymphoma, accounting for 2% to 3% of NHL. The disease typically presents as a large mediastinal mass in patients in the AYA age range, with an increased incidence in females. Although previously considered a subtype of diffuse large B-cell lymphoma, PMBCL is now recognized as its own clinical and molecular/pathologic entity that shares many similarities with Hodgkin lymphoma. Recent genomic characterization of PMBCL has been performed using whole-exome sequencing (Mottok et al., Blood 2019; Chapuy et al., Blood 2019). These studies reported recurrent alterations in components of JAK/STAT and NF-κB signaling as well as genetic mechanisms of immune evasion similar to that observed in cHL including alterations in B2M, CD274, and CIITA. In addition, cases were observed to have high tumor mutational burden and an apolipoprotein B mRNA editing catalytic polypeptide-like (APOBEC) mutation signature, which may confer sensitivity to PD-1 blockade. In summary, recent genomic findings in HL and PMBCL demonstrate molecular similarities between these lymphoma subtypes and define the genetic basis for immune evasion sensitivity to PD-1 blockade. This has translated to national clinical trials for AYAs with HL and PMBCL. A randomized phase III trial of nivolumab + AVD vs. brentuximab + AVD for children and adults with advanced stage HL is currently ongoing (NCT03907488). In PMBCL, a randomized phase III trial evaluating nivolumab in combination with standard therapy for upfront disease is in development and will include pediatric and adult patients across NCTN sites. Citation Format: Lisa G. Giulino-Roth. Genomics of common AYA lymphomas: Hodgkin lymphoma and primary mediastinal B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA09.