Abstract: DLCBL has recently been classified into genetically defined subtypes based on groupings of particular genetic lesions (Chapuy et al, Nat Med 2018; Schmitz et al, NEJM 2018). One predominant cluster, C3 or EZB, is defined by mutations in the chromatin modifying genes EZH2, KMT2D, and CREBBP as well as alterations in BCL2 including mutations and/or translocation of BCL2 to the IgH enhancer. Since tumors in this cluster are likely dependent on both EZH2 and BCL2, and these oncogenes carry out their effects through distinct mechanisms and pathways, targeting both of these oncogenes is a rational therapeutic approach. We hypothesized that EZH2 inhibition and BCL2 inhibition would be synergistic in DLBCL with characteristics of the C3/EZB cluster. To test this, we evaluated the EZH2 inhibitor tazemetostat and the BCL2 inhibitor venetoclax in DLBCL cells, 3D lymphoma organoids, and patient derived xenografts. To assess the effect of combination therapy with tazemetostat and venetoclax, we administered each drug alone and the combination in a panel of DLBCL cell lines, including cells with and without EZH2 mutation and BCL2 translocation. In DLBCL cells with both a BCL2 translocation and EZH2 mutation, the combination resulted in increased killing compared to either drug alone (Figure 1, SUDHL6 (p & lt;0.005), WSU-DLCL2 (p & lt;0.005), and OCI-LY1 (p & lt;0.005)). In contrast, in cells with WT EZH2 and no BLC2 translocation, the effect of the combination was not different than either drug alone. To evaluate for synergy, cells were exposed to increasing doses of each drug alone and the combination. The combined response was evaluated using the Chou-Talalay method. Synergy between tazemetostat and venetoclax was observed in SUDLH-6 (CI value 0.03), WSU-DLCL2 (CI 0.26) and OCI-LY1 (CI 0.06) but not in Farage and LY7 both of which have WT EZH2 and no BCL2 translocation. Since cell lines in suspension do not reflect lymph node architecture, we developed a 3D lymphoma "organoid" culture system that consists of extracellular matrix, lymphoma cells, and stromal cells (Tian et al, Biomaterials 2015; Beguelin et al, Nat Commun 2017). GCB-DLBCLs express integrin αvβ3 that may bind to RGD peptides or vitronectin in tumor extracellular matrix. Based on this, we developed matrix metalloproteinase (MMP)-degradable 3D lymphoma hydrogels functionalized with RGD peptides. To generate organoids, we homogeneously encapsulated DLBCL cells (40,000/gel) in 10 µL hydrogel droplets fabricated in individual wells of a 96-well plate. We established two organoid systems to evaluate response to tazemetostat/venetoclax combination therapy: 1) OCI-LY1 organoids; 2) patient derived xenograft (PDX) organoids. The PDX organoids were generated from a patient tumor after propagation in NSG mice. The PDX tumor harbors both a BCL2 translocation and EZH2 mutation. Organoid viability was evaluated using immunofluorescence for calcein AM (live cells) and ethidium homodimer (dead cells) as well as flow cytometry. In both OCI-Ly1 organoids and PDX organoids, tazemetostat and venetoclax had minimal activity as single agents, however the combination resulted in significant cell killing (Figure 2). To investigate potential mechanisms of synergy, we evaluated RNA-seq profiles of a panel of DLBCL cell lines (n=26) treated with vehicle vs. EZH2 inhibitor (Brach et al, Mol Cancer Ther 2017). Preliminary data suggest that EZH2 inhibition induces expression of pro-apoptotic proteins genes including BCL2L11 (Wilcoxon p=0.01), BAD (p=0.02), BMF (p & lt;0.01), BCL2L13 (p=0.02), and BCL2L14 (p & lt;0.01). BCL2 inhibition with venetoclax may be further enhancing pro-apoptotic signals and lymphoma cell death, especially in C3/EZB DLBCL tumors with dependence on BCL2. In summary, using novel model systems, we have demonstrated that BCL2 inhibition combined with EZH2 inhibition results in synergistic anti-tumor effect that is anticipated to be especially effective as precision therapy for the newly identified cluster 3/EZB DLBCL subtype. A clinical trial of this combination is currently in development. Disclosures Melnick: Janssen: Research Funding; Constellation: Consultancy; Epizyme: Consultancy. Roth:Janssen: Consultancy; ADC Therapuetics: Consultancy; Merck: Membership on an entity's Board of Directors or advisory committees.

Abstract: Abstract 899 While the majority of children with Burkitt lymphoma (BL) are cured with conventional chemotherapy, outcome for patients with relapsed disease is poor (overall survival 〈 30%). Cure rates are lower in developing countries, which bear the brunt of this disease, including the endemic form in sub-Saharan Africa. Targeted therapy would be of benefit to all patients with BL to improve outcomes and decrease the reliance on conventional toxic chemotherapy. In order to develop such therapies a comprehensive understanding of the BL cancer genome is needed. The hallmark of BL is the translocation of MYC to either the immunoglobulin heavy or light chains. In contrast to adult cases, pediatric tumors often have multiple additional cytogenetic abnormalities, the consequences of which are less well understood. In order to characterize the genomic landscape of pediatric BL and to investigate potential therapeutic targets, we performed targeted massively parallel sequencing on a cohort of primary pediatric BL samples. Large genomic studies in rare tumors such as pediatric BL have been limited by the need for frozen tissue. In the current study we utilized a platform that has been adapted for genomic analysis of formalin-fixed, paraffin-embedded (FFPE) tissue, which allows the use of archival FFPE cases (Lipson, et al Nat Med. 2012 Feb 12;18(3):382-4). Targeted genomic sequencing was performed on 29 pediatric BL cases evaluating 3,230 exons of 182 cancer related genes and 37 introns from 14 genes often rearranged in cancer. Tumor samples were collected at diagnosis from patients in the US, Brazil, and Kenya and included the sporadic (n=24), endemic (n=3) and HIV associated (n=2) forms of disease. At an average coverage of 653-fold, 89% of cases were found to have at least one genetic alteration. The number of observed alterations correlated with EBV status. EBV negative cases demonstrated significantly more genomic alterations than EBV-positive cases. Among the EBV(–) cases, 13/15 had 〉 1 alteration compared with 2/13 EBV(+) cases with 〉 1 alteration (p 〈 0.001). This is consistent with a tumorigenic role of EBV in BL. The most common mutations observed were in MYC (58.6%) and p53 (41.4%). MYC mutations spanned the coding region and included hot spots previously documented in lymphomas. Cases with MYC mutations also demonstrated MYC translocations, confirming that mutations may functionally cooperate with translocation to promote MYC-mediated oncogenesis. Novel recurrent alterations were identified in the chromatin remodeling gene, ARID1A as well as the anti-apoptotic gene, MCL1. ARID1A is a member of the SWI/SNF family of complexes that regulate chromatin structure and has been implicated as a tumor suppressor in a variety of solid tumors, but has not yet been described in BL. We identified truncating mutations in 5/29 cases (17.2%), including one case that had a secondary mutation in SWF5, also a member of the SWI/SNF family. Immunohistochemistry for ARID1A demonstrated decreased expression in cases where the truncating mutation was upstream of the antibody epitope. MCL1 is a member of the Bcl2 family of apoptotic proteins and has been implicated as an oncogene in lymphomas. Of the 29 cases sequenced, 6 (20.7%) had alterations in the MCL1 pathway including amplification of MCL1 (n=5) and point mutation in FBWX7, a ubiquitin ligase that targets MCL1 for degradation (n=1). Amplification of MCL1 was confirmed by FISH. In an independent cohort, 5/17 (29.4%) cases demonstrated MCL1 amplification by FISH. MCL1 protein expression, as evaluated by immunohistochemistry, was increased in 19/40 cases including the cases known to have MCL1 amplification. This study is the first genomic analysis of BL using FFPE tissue and demonstrates the feasibility of next generation sequencing of rare lymphomas using archival FFPE tissue. Our findings highlight the potential role of anti-apoptotic and chromatin remodeling genes in BL pathogenesis. Disclosures: Wang: Foundation Medicine: Employment, Equity Ownership. Cronin:Foundation Medicine: Employment, Equity Ownership. Palmer:Foundation Medicine: Employment, Equity Ownership. Yelensky:Foundation Medicine: Employment, Equity Ownership. Stephens:Foundation Medicine: Employment, Equity Ownership.

Abstract: 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.

Abstract: 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 (age & lt;18), 9 adolescents and young adults (AYA, age 18-40), and 6 older adults (age & gt;40). 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 ( & gt;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 & lt;0.0001 in Peds/AYA; R 2 =0.82; p=0.002 in older adults). We observed a higher mutation rate in Pediatric/AYA cases compared to older adults (p=0.01), which is consistent with the higher mutational burden observed in this age group. By estimating the SBS1- and SBS5-based molecular time for large chromosomal gains and converting relative estimates to absolute time, we are able to estimate the age in years at the time of the first multi-chromosomal gain event. We observed that the first multi-chromosomal gain in cHL is often acquired several years before the diagnosis/sample collection: median latency of 19.5 (range 12-27) and 5.6 (range 1.8-16) years in older adults and pediatric/AYA patients respectively. 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.

Abstract: 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 & gt;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 & lt;12 months after frontline therapy. The median number of salvage regimens received pre-ASCT was 2 (range 1-7). Forty-two patients (81%) received BV as part of a pre-ASCT salvage regimen, 4 (8%) of whom did not receive BV in the regimen immediately prior to ASCT. Radiation therapy was used pre or post-ASCT in 16 patients (31%). Prior to ASCT, 40 patients (77%) were PET-negative (Deauville 1-3), 11 (21%) were PET-positive (Deauville 4-5), and response in 1 (2%) was unknown. 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.

Abstract: 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.

Abstract: TPS7567 Background: High risk for relapse is observed in cHL patients (pts) with SER to initial chemotherapy and organ toxicities may be higher following dose intensification. Methods: The phase 2 KEYNOTE-667 study will enroll 440 pts aged 3 to 17 (children) or 18 to 25 years (young adults) with newly diagnosed, confirmed stage IA, IB, or IIA cHL without bulky disease (Group 1 [low-risk]) or stage IIEB, IIIEA, IIIEB, IIIB, IVA, or IVB cHL (Group 2 [high-risk] ); measurable disease; and performance status per Lansky Play-Performance Scale ≥50 (age ≤16 years) or Karnofsky score ≥50 (age 〉 16 years). Pts will receive induction with doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD; Group 1) or vincristine, etoposide/etoposide phosphate, prednisone/prednisolone, doxorubicin (OEPA; Group 2) for 2 cycles, then early response assessment by PET/CT/MRI. Pts with rapid early response (Deauville score 1-3) will receive standard therapy. Pts with SER (Deauville score 4-5) will receive consolidation with pembro 2 mg/kg Q3W up to 200 mg (children) or 200 mg Q3W (young adults) plus 2 cycles AVD (Group 1) or 4 cycles cyclophosphamide, vincristine, prednisone/prednisolone, dacarbazine (COPDAC-28; Group 2). PET/CT for late response assessment (LRA) will be performed after consolidation. After LRA, Group 1 pts with SER and Group 2 pts with Deauville score 4-5 will receive radiotherapy (RT). All pts will receive maintenance with pembro Q3W concomitantly with RT. Pembro will continue up to 17 administrations, with an option to stop after 24 weeks due to CR, or until progression, unacceptable toxicity, or withdrawal. The primary endpoint is ORR per Cheson 2007 IWG criteria by group in SER pts. Secondary endpoints are SERs with PET negativity after consolidation, 2-yr event-free survival (EFS), OS, and RT frequency and details by group, RERs with PET negativity after ABVD induction, 3-yr EFS by investigator, and OS by risk group, and serum TARC levels at screening in SERs by risk group. ORR with 95% CI will be estimated by Clopper-Pearson method. EFS and OS will be estimated by Kaplan-Meier method. Safety will be assessed in all treated pts. Clinical trial information: NCT03407144.