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Introduction: HIV-associated diffuse large B-cell lymphoma (HIV-DLBCL) is a significant complication in HIV-positive individuals, presenting unique clinical challenges and a poorer prognosis compared to patients with de novo DLBCL. Epstein-Barr Virus (EBV) status is a critical factor influencing the pathogenesis, clinical behavior, and therapeutic responses in these lymphomas. Plasmablastic lymphomas (PBL) have shared some overlapping genomic abnormalities with HIV-DLBCL including MYC rearrangements. This study aims to elucidate the differences in the genetic profiles and pathway activations between EBV(+) and EBV(-) HIV-DLBCL through comprehensive genomic profiling. Methods: Cases of DLBCL were identified in patients living with HIV from a multi-institute cooperative group. Cases of Burkitt lymphoma and PBL were excluded. We performed whole exome sequencing (WES) on 106 tumors of HIV-DLBCL and RNA sequencing on 39 tumors and mapped the reads to hg38 reference genome using BWA and STAR, respectively. EBV status of cases was determined by in situ hybridization and/or reads mapped on EBV genome in sequencing data. Somatic mutations were identified using variant callers (Strelka2, Mutect2) with WES data. For those mutated genes in COSMIC, allele frequency cutoff was set at 0.03. Mutations with an artifact score larger than 0.7 in SOBDetector and those located in segmental duplications were further removed. They were annotated with Ensembl Variant Effect Predictor, manually checked, and plotted with maftools. RNAseq read counts aligned to each gene were obtained by featureCounts and DEseq2 was used for differential expression (DE) analysis between EBV positive and negative groups. Pathway analysis with the DE genes was performed using GSEA. FISH analysis was performed using MYC, BCL2, and BCL6 break-apart probes. Results: There were 41 cases of EBV(+) HIV-DLBCL and 65 cases of EBV(-) HIV-DLBCL. FISH of EBV(+) HIV-DLBCL identified 42% (3/7) with MYC rearrangement (MYCr), none with BCL2 rearrangements (BCL2r) (0/6), and 33% (2/6) positive for BCL6 rearrangement (BCL6r) with 1 tumor having a MYCr/BCL6r. For EBV(-) HIV-DLBCL, there were 35% with a MYCr (6/17), 18% with BCL2r (2/11), and 27% with a BCL6r (3/11) with 2 tumors with MYCr/BCL6r, and 1 tumor with MYCr/BCL2r/BCL6r. The most frequent mutations seen in EBV(+) HIV-DLBCL were STAT3 (32%), DDX3X (22%), and TP53 (15%). For EBV(-) HIV-DLBCL, the most frequent mutations were KMT2D (25%), TP53 (23%), GNA13 (22%), and TET2 (22%). Pathways that were most commonly associated with EBV(+) HIV-DLBCL were JAK/STAT (42%) (STAT3, JAK1) and NF-kB (39%)(DDX3X, TNFAIP3) while EBV(-) HIV-DLBCL was associated with epigenetic modifiers (62%)(KMT2D, TET2, EP300), JAK/STAT (45%)(SOCS1, JAK3) and PI3K/mTOR (40%)(GNA13, MTOR). Significant disparities between EBV(+) and EBV(-) were KMT2D (24% vs 50%, P=0.0025), TET2 (10% vs 26%, P=0.008), SOCS1 (29% vs 39%, P=0.04), DDX3X (15% vs 5%, P=0.03), and GNA13 (5% vs 24%, P=0.001). RAS mutations (KRAS, NRAS) were infrequent in HIV-DLBCL (EBV(+), 12%; EBV(-), 6%), in contrast to prior studies of PBL. Gene expression profiling showed enrichment of MYC targets, oxidative phosphorylation, and the mTORC pathway in EBV(+) as compared to EBV(-) HIV-DLBCL. Conclusion: EBV(+) HIV-DLBCL show a distinct mutational profile compared to EBV(-) HIV-DLBCL, with EBV(+) cases showing frequent mutations with STAT3 and DDX3X in contrast to EBV(-) cases with frequent mutations associated with epigenetic modification pathways (KMT2D, TET2). These genetic profiles underscore the need for tailored therapeutic approaches depending on EBV status. Although the analysis is limited and requires confirmation in a larger cohort, this is the largest study at present evaluating these tumors.

Introduction Peripheral T-cell lymphoma (PTCL) encompasses a heterogeneous group of clinically aggressive entities. ~30% of cases cannot be further classified and are designated as PTCL - not otherwise specified (NOS). We delineated two novel molecular subgroups among PTCL-NOS with distinct clinical and biological features: PTCL-GATA3 which is characterized by high expression of GATA3, a master regulator of T-helper-2-cell (TH2) differentiation, and its target genes (i.e., CCR4, IL4, IL13) and PTCL-TBX21 which is characterized by high expression of TBX21, a master regulator of TH1 differentiation, and its target genes (i.e., CXCR3, IFNG), with the GATA3 subtype showing significantly worse clinical outcomes (overall survival). Nonsense mutations in CCR4 are found predominately in PTCL-GATA3, often within the cytoplasmic domain and gain-of-function in nature. Methods Mutation and copy number data were generated from whole exome sequencing (TFH phenotype excluded following LLMPP pathology review; n = 153). Single nucleotide variants were determined using Mutect2 and Varsvan2 using in-house filtering. Copy number (CN) profiles were generated using CNVkit. Four CCR4 cytoplasmic domain mutants and controls (cytoplasmic domain knockout [CDKO], wild-type CCR4 overexpression (CCR4-WT), and empty vector [EV]) were transduced into healthy-donor CD4+ T cells (n = 2). Polarization studies were generated using TH1- or TH2-associated cytokine exposure. Surface plasmon resonance was used to determine the binding affinity of CCR4 proteins to CCL17 and CCL22. In silico docking studies were done through Schrödinger's BioLuminate. Antibody-dependent cell-mediated cytotoxicity (ADCC) assay was performed through co-incubation of NK92 (CD16 and CD56) and CCR4 edited primary CD4+ T-cells (stained with calcein AM) for 2 hours at a 5:1 NK:T cell ratio by 7-AAD flow cytometry. Results PTCL-GATA3 had a higher frequency of CN aberrations (e.g. TP53, PTEN CN loss) and a more aberrant genome (p < 0.001) than PTCL-TBX21, including mutations with genes associated with DNA repair and damage (TP53, ATM) and T-cell differentiation (CCR4). CCR4 mutations were virtually mutually exclusive with TP53 mutations, and these cases showed higher CCR4 mRNA and protein expression than CCR4-WT cases (p < 0.05). Primary CD4+ T-cells with ectopic expression of the CCR4 variants, especially CCR4-Q330X, did not require CD3/CD28 for proliferation, bypassing CD3/CD28 ligation for T-cell activation. In addition, proliferation rates were similar between TH2-conditional media and normal media, but proliferation and TBX21 expression in TH1-conditional media were inhibited, implying a novel role of CCR4 in T-cell receptor (TCR) activation and TH-differentiation. CCR4 variant proteins showed higher binding affinity for their natural ligands, CCL22 and CCL17, with mutants exhibiting tighter binding to both ligands (1.5- to 3-fold decrease in KD) by surface plasmon resonance and in silico modeling studies. The in silico models demonstrated mutant CCR4 bound mogamulizumab tighter than CCR4-WT, consistent with a higher ADCC-induced apoptosis rate, highest with CCR4-Q330X mutation. CCR4-Y331X exhibited a worse docking score compared to CCR4-WT and a lower ADCC-induced apoptosis rate. Conclusions Our findings reveal distinct genetic characterizations including aberrant TP53 and PI3K signaling and CCR4 mutations in PTCL-GATA3. These CCR4 mutations are shown to have a key role in PTCL-GATA3 pathogenesis by dysregulating TH-differentiation and aberrantly activating the TCR. CCR4 mutation promotes the TH2 phenotype by blocking TH1 differentiation. CCR4 mutant proteins demonstrated an increased binding affinity towards its known ligands, CCL17 and CCL22, and mogamulizumab, consistent with previous studies exhibiting mogamulizumab efficacy against CCR4 mutations. However, the most frequent CCR4 mutant, Y331X, showed worse binding compared to other CCR4 mutants or CCR4-WT, a mutation that showed Mogamulizumab-resistance in a prior CTCL study as well as in an in vitro ADCC assay. The results emphasize aberrant pathways implicated by genomic and clinical evaluation of two novel PTCL subtypes, as well as support the novel role of mutant CCR4 in enhancing T-cell lymphomagenesis and T-helper differentiation, highlighting multiple therapeutic targets.

Introduction Plasmablastic lymphoma (PBL) is a rare aggressive subtype of lymphoma. It is more common in HIV positive patients, and is often associated with Epstein-Barr virus (EBV) and MYC rearrangements. PBL is characterized by markers of plasmacytic differentiation including CD138 and MUM1/IRF4, and loss of B-cell markers such as CD20, which suggests a cell of origin similar to that of multiple myeloma (MM) or activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL). The standard of care for PBL is CHOP-based chemotherapy but this is frequently not curative. Other than EBV-negative status, which defines a high-risk group, no other biological features have been discovered that are associated with outcomes. Methods Here, we provide a comprehensive analysis of the genomic and transcriptomic landscape of PBL using a collection of 176 exomes and genomes (n=55 new, contributed by Lymphoma/Leukemia Molecular Profiling Project [LLMPP] sites and centrally reviewed by the LLMPP pathology panel [WHO 2016]; n=121 previously published [PMID: 33225311, 33951889, 34465776, 34714908]), 65 transcriptomes (n=44 new; n=21 published) and 37 in situ single cell transcriptomic profiles (CosMx Spatial Molecular Imager) from archival diagnostic tissue biopsies. Somatic mutations (SNVs/Indels) were identified using an ensemble of four variant callers (Strelka2, Lofreq, Mutect2, SAGE). Salmon and DESeq2 were used to identify differentially expressed genes. This multi-omics dataset was compared to other known B-cell malignancies including 92 MM, 238 Burkitt lymphomas (BL), and 208 DLBCLs. Cell segmentation of the CosMx data was performed using Baysor and downstream analyses were conducted using the Seurat workflow. For functional validation, cell viability assays (CellTiter-Glo Luminescent Assay) were conducted in the PBL cell line, PBL-1, and 4 control DLBCL cell lines. Results Within the cohort, 63% (106/169) of primary PBL cases were EBV-positive (EBV+) and 57% (82/144) harbored MYC rearrangements (MYC+). When stratifying patients by EBV and MYC rearrangement status, mutations in different genes were enriched in each group: STAT3 occurred in 55% (29/53) of EBV+/MYC+ PBLs; NOTCH1 in 19% (7/36) of EBV+/MYC-; MYC in 24% (6/25) and TP53 in 40% (10/25) of EBV-/MYC+ tumors; TET2 was mutated in 38% (9/24) and KRAS in 25% (6/24) of EBV-/MYC- PBLs. EBV-/MYC+ tumors represented a subgroup of patients with dismal outcomes. The 2-year overall survival (OS) of these patients was 22% compared to 61%, 78%, and 54% of EBV+/MYC+, EBV+/MYC-, and EBV-/MYC- PBL patients, respectively (log-rank test p-adj. < 0.05). Consistent with previous studies, we observed recurrent mutations affecting JAK-STAT (STAT3, SOCS1, SOCS3, DUSP2) in 41% of cases and RAS-RAF (NRAS, KRAS, BRAF) signaling in 38% of all PBLs. In contrast to ABC-DLBCL, very few mutations occurred in the NF-κB signalling pathway. Accordingly, PBL showed marked downregulation of genes involved in B-cell receptor and NF-κB signaling. Consistent with the lack of reliance on these pathways, functional analyses in PBL-1 showed resistance to treatment to ibrutinib as well as PI3K- and MALT1-inhibitors. Moreover, constitutive expression of the IκBα super-repressor did not affect viability of PBL-1 cells. In situ single cell transcriptomic data analyses showed a sparse tumor microenvironment (TME) in PBL, with the primary non-malignant cell types being, on average, 10% macrophages, 7% NK/T cells, and 4% stromal cells of all represented cell types. EBV+/MYC+ tumors displayed a more abundant immune cell population with higher levels of macrophages (median 12%) and NK/T cells (median 9%), whereas EBV-/MYC- tumors were composed of the lowest levels of immune cells (median 4% macrophages and 1% NK/T cells). With respect to malignant cell phenotypes, we noted a novel population of SPP1-expressing cells that were enriched in EBV+/MYC+ tumors and a population of CD44-expressing cells that were enriched in EBV+/MYC- tumors. Conclusion Here we present novel subgroups of PBL with unique biology and clinical outcomes. We show that PBL does not rely on B-cell receptor signaling, instead relying on potentially targetable vulnerabilities in JAK-STAT, NOTCH, and RAS-RAF signaling according to EBV positivity and MYC rearrangement status. Lastly, we provide insight into the TME of PBL and demonstrate a novel population of SPP1 and CD44 expressing malignant cells in EBV+ tumors.

Abstract Disclosure: D. Ferone: Consulting Fee; Self; Camurus AB, Ipsen, Novartis-AAA, Recordati. Grant Recipient; Self; Camurus AB, Ipsen, Novartis-AAA, Recordati. Research Investigator; Self; Camurus AB. J.L. Spencer-Segal: Advisory Board Member; Self; Camurus AB. Research Investigator; Self; Camurus AB. G. Fidan Yaylali: None. M. Doknic: Speaker; Self; Pfizer, Inc., Novo Nordisk, Merck, Sandoz. E. Isaeva: None. A. Dreval: None. A. Gilis-Januszewska: None. E. Gezer: None. I. Bancos: Advisory Board Member; Self; Diurnal, Spruce, Neurocrine, Adrenas, Sparrow, Corcept, HRA Pharmaceuticals, Recordati, Xeris, AstraZeneca, Novo Nordisk. Grant Recipient; Self; Recordati, HRA Pharmaceuticals. L. Katznelson: Advisory Board Member; Self; Camurus AB, Recordati. M. Harrie: Employee; Self; Camurus AB. Stock Owner; Self; Camurus AB. A. Svedberg: Employee; Self; Camurus AB. Stock Owner; Self; Camurus AB. A.M. Pedroncelli: Employee; Self; Camurus AB. Stock Owner; Self; Camurus AB. F. Tiberg: Employee; Self; Camurus AB. Stock Owner; Self; Camurus AB. Background: Acromegaly is a rare, serious disorder characterized by overproduction of growth hormone (GH) and insulin-like growth factor 1 (IGF-1). There is a need for new, convenient therapies that provide effective disease control. CAM2029 is a novel subcutaneous octreotide depot designed for convenient once-monthly self-administration (ready-to-use syringe or injection pen). In a 24-week, Phase 3 trial (ACROINNOVA 1), CAM2029 was superior to placebo at attaining IGF-1 response (≤upper limit of normal [ULN] at week 22/24: 72.2% vs 37.5%; P=0.0018) in patients with acromegaly previously controlled under standard of care (SoC; octreotide long-acting repeatable [LAR]/lanreotide Autogel). Here, we report interim data from another Phase 3 trial of CAM2029 (ACROINNOVA 2; NCT04125836), focusing on the “New to CAM2029” subgroup of patients whose acromegaly was either controlled or uncontrolled under SoC. Methods: This 52-week, Phase 3, open-label trial (with a 52-week extension) enrolled patients who had received CAM2029 or placebo in ACROINNOVA 1 (reported separately) and “New to CAM2029” patients who were adequately or inadequately controlled (IGF-1 ≤2x ULN) on a stable dose of octreotide LAR/lanreotide Autogel for ≥3 months (data reported here). In the main part of the trial, new patients received once-monthly CAM2029 20 mg for up to 52 weeks. The primary endpoint was adverse events (AEs). Secondary endpoints included (i) the proportion of patients with IGF-1 ≤1x ULN (weeks 50/52 mean), (ii) the change from baseline in IGF-1, and (iii) the combined IGF-1 and GH response. Baseline values reflected SoC treatment. Data cutoff: May 23, 2023. Results: Eighty-one new patients were enrolled (mean age 51.8 years; 59.3% female). No new or unexpected safety signals were observed. The most common treatment-emergent AEs were injection site erythema (n=22) or swelling (n=13), and headache (n=12). AEs were mostly mild (grade 1/2); there were no serious or Grade 3 treatment-related AEs attributed to CAM2029. IGF-I response increased from 14.8% at baseline SoC (12/81) to 33.3% (20/60 patients with available data) at weeks 50/52. The estimated increase in IGF-1 response (linear probability model) from baseline at week 52 was 21.9% (95% CI: 9.6, 34.1). Biochemical control for both IGF-1 and GH improved over time: 30.0% of patients (18/60) had a combined IGF-1 and GH response at the end of the main part of trial, vs 11.7% (7/60) at baseline. Conclusion: CAM2029 was well tolerated with a safety profile consistent with that of SoC. Biochemical control rates improved from SoC baseline over 52 weeks of CAM2029 treatment in the “New to CAM2029” patients. These findings reinforce the efficacy and safety of CAM2029 in patients with acromegaly, including patients with uncontrolled disease with current SoC treatment (octreotide LAR/lanreotide Autogel). Presentation: 6/1/2024

Abstract Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements (“double hit”; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

OBJECTIVE Traditionally, resection of nondominant hemisphere brain tumors was performed under general anesthesia. An improved understanding of right-lateralized neural networks has led to a paradigm shift in recent decades, where the right or nondominant hemisphere is no longer perceived as "functionally silent." There is an increasing interest in awake brain mapping for nondominant hemisphere resections. The objective of this study was to perform a comprehensive review of the existing brain mapping paradigms for patients with nondominant hemisphere gliomas undergoing awake craniotomies. METHODS In accordance with PRISMA guidelines, systematic searches of the Medline, Embase, and American Psychological Association PsycInfo databases were undertaken from database inception to July 1, 2023. Studies providing a description of the intraoperative mapping paradigm used to assess cognition during an awake craniotomy for resection of a nondominant hemisphere glioma were included. RESULTS The search yielded 1084 potentially eligible articles. Thirty-nine unique studies reporting on 788 patients were included in the systematic review. The most frequently tested cognitive domains in patients with nondominant hemisphere tumors were spatial attention/neglect (17/39 studies, 43.6%), speech-motor/language (17/39 studies, 43.6%), and social cognition (9/39 studies, 23.1%). Within the frontal lobe, the highest number of positive mapping sites was identified for speech-motor/language, spatial attention/neglect, dual tasking assessing motor and language function, working memory, and social cognition. Within the parietal lobe, eloquence was most frequently found upon testing spatial attention/neglect, speech-motor/language, and calculation. Within the temporal lobe, the assessment of spatial attention/neglect yielded the highest number of positive mapping sites. CONCLUSIONS Cognitive testing in the nondominant hemisphere is predominantly focused on evaluating two domains: spatial attention/neglect and the motor aspects of speech/language. Multidisciplinary teams involved in awake brain mapping should consider testing an extended range of functions to minimize the risk of postoperative deficits and provide valuable information about anatomo-functional organization of cognitive networks.