Pittaluga, Stefania

Abstract With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

Abstract Introduction: Plasmablastic lymphoma (PBL) is an aggressive B-cell lymphoma that predominantly occurs in patients with HIV or other causes of immunodeficiency. Frequent infection by the Epstein-Barr virus (EBV) and MYC translocations have been described as major features contributing to the pathogenesis of PBL. Prior studies examining the genetic landscape of PBL have largely relied on targeted capture-based sequencing approaches. As such, the molecular features of PBL remain to be fully explored. Here, we provide a comprehensive description of the genomic landscape of PBL using whole-genome and whole-exome sequencing to identify commonly perturbed pathways. Method: Archival diagnostic fresh frozen and formalin fixed paraffin embedded tissue biopsies from 58 PBL tumours were accrued from Lymphoma/Leukemia Molecular Profiling Project (LLMPP) sites, including 15 tumours from Ramis-Zaldivar et al., Haematolgica 2021. MYC rearrangements were identified by break-apart fluorescent in situ hybridization (FISH) and rearrangement partner was determined in a subset of tumours from capture or genome sequencing data using structural variant callers Manta, GRIDSS, and Delly. High confidence somatic mutations (SNVs/Indels) were identified in data from whole-genome (n=5) or whole-exome (n=53) sequencing through an ensemble voting approach utilizing four variant callers (Strelka2, Lofreq, Mutect2, SAGE). Mutation frequencies in known lymphoma-related genes were compared to activated B-cell (ABC)-DLBCL (Schmitz et al., NEJM 2018), as the closest tumour entity in terms of putative cell-of-origin differentiation stage, to identify differences in genetic aberrations. Candidate somatic copy number alterations (CNAs) were identified from exome and genome sequencing data, using CopywriteR and ControlFREEC, respectively, and high-confidence CNAs were determined using GISTIC2.0. Results: Within the study cohort, 81% of patients were male with a median age of 59 years (range 11-88). HIV and EBV statuses were available for 47% of patients and 95% of tumours, respectively, with 49% (13/27) of the patients being HIV+ and 69% (38/55) of tumours being EBV+. MYC rearrangement was observed in 60% (35/58) of PBLs, with IGH as the partner gene in 88% (21/24) of tumours. The most frequently mutated genes were STAT3 (38%), TP53 (22%), NRAS (21%), and TET2 (16%), consistent with previous studies, however novel mutations were seen in DUSP2 (21%), KLHL6 (16%), and BHLHE41 (16%). Recurrent CNAs included amplifications in 1q, whole gains of 7, 8q24, 11p12 and deletions affecting 4p16, 5p15, 10q11.22. While the mutational landscapes were similar between samples with and without a MYC translocation, the MYC-translocated PBLs showed more frequent amplification of 1q32.1. When stratifying by EBV status, STAT3 and SOCS1 mutations were more frequent in EBV-positive tumours, whereas TP53, TET2, KRAS, and MMRN2 mutations were associated with EBV-negativity. In comparison to ABC-DLBCL, PBLs were significantly enriched in STAT3 and NRAS mutations, and lacked common mutations affecting the NF-κB pathway (eg. MYD88, CD79B, and NFKBIZ 3' UTR mutations). Mutations in genes that are frequently mutated in ABC-DLBCLs, such as those associated with plasma cell differentiation (eg. PRDM1) or a memory B-cell fate (eg. TBL1XR1), were also not mutated in PBLs. Finally, genetic alterations associated with immune evasion, such as deletion of MHC I and II and mutations in B2M, CIITA, and CD58, were rarely observed. Conclusion: These data present a comprehensive overview of the genomic landscape of PBLs in a large cohort. We show frequent mutations involving the JAK-STAT and MAPK pathways, wherein the genetic landscape can be differentially characterized by EBV status and MYC rearrangement status. We show that PBLs are genetically distinct from ABC-DLBCLs, with absence of mutations in genes affecting the NF-κB pathway, immune evasion, and driving a memory B-cell fate. Disclosures Slack: Seagen: Consultancy, Honoraria. Raess: Scopio Labs: Research Funding. Holte: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Nordic: Membership on an entity's Board of Directors or advisory committees; Nanovector: Membership on an entity's Board of Directors or advisory committees, Other: lectures honorarias; Novartis: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Savage: Servier: Consultancy, Honoraria; Roche: Research Funding; BMS: Consultancy, Honoraria, Other: Institutional clinical trial funding; Astra-Zeneca: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Other: Institutional clinical trial funding; AbbVie: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Takeda: Other: Institutional clinical trial funding; Beigene: Other: Institutional clinical trial funding; Genentech: Research Funding. Steidl: Seattle Genetics: Consultancy; Curis Inc.: Consultancy; Bayer: Consultancy; Epizyme: Research Funding; Trillium Therapeutics: Research Funding; AbbVie: Consultancy; Bristol-Myers Squibb: Research Funding. Rimsza: NanoString Technologies: Other: Fee-for-service contract. Morin: Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Epizyme: Patents & Royalties. Scott: Abbvie: Consultancy; AstraZeneca: Consultancy; Rich/Genentech: Research Funding; BC Cancer: Patents & Royalties: Patent describing assigning DLBCL COO by gene expression profiling--licensed to NanoString Technologies. Patent describing measuring the proliferation signature in MCL using gene expression profiling. ; Incyte: Consultancy; Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling..

Abstract Techniques that enable longitudinal tracking of cell fate after myocardial delivery are imperative for optimizing the efficacy of cell-based cardiac therapies. However, these approaches have been underutilized in preclinical models and clinical trials, and there is considerable demand for site-specific strategies achieving long-term expression of reporter genes compatible with safe noninvasive imaging. In this study, the rhesus sodium/iodide symporter (NIS) gene was incorporated into rhesus macaque induced pluripotent stem cells (RhiPSCs) via CRISPR/Cas9. Cardiomyocytes derived from NIS-RhiPSCs (NIS-RhiPSC-CMs) exhibited overall similar morphological and electrophysiological characteristics compared to parental control RhiPSC-CMs at baseline and with exposure to physiological levels of sodium iodide. Mice were injected intramyocardially with 2 million NIS-RhiPSC-CMs immediately following myocardial infarction, and serial positron emission tomography/computed tomography was performed with 18F-tetrafluoroborate to monitor transplanted cells in vivo. NIS-RhiPSC-CMs could be detected until study conclusion at 8 to 10 weeks postinjection. This NIS-based molecular imaging platform, with optimal safety and sensitivity characteristics, is primed for translation into large-animal preclinical models and clinical trials.

Abstract Rare cases of histiocytic and dendritic cell (H/DC) neoplasms have been reported in patients with follicular lymphoma (FL), but the biologic relationship between the 2 neoplasms is unknown. We studied 8 patients with both FL and H/DC neoplasms using immunohistochemistry, fluorescence in situ hybridization (FISH) for t(14;18), and polymerase chain reaction (PCR)/sequencing of BCL2 and IGH rearrangements. There were 5 men and 3 women (median age, 59 years). All cases of FL were positive for t(14;18). The H/DC tumors included 7 histiocytic sarcomas, 5 of which showed evidence of dendritic differentiation, and 1 interdigitating cell sarcoma. Five H/DC tumors were metachronous, following FL by 2 months to 12 years; tumors were synchronous in 3. All 8 H/DC tumors showed presence of the t(14;18) either by FISH, or in 2 cases by PCR with the major breakpoint region (MBR) probe. PCR and sequencing identified identical IGH gene rearrangements or BCL2 gene breakpoints in all patients tested. All H/DC tumors lacked PAX5, and up-regulation of CEBPβ and PU.1 was seen in all cases tested. These results provide evidence for a common clonal origin of FL and H/DC neoplasms when occurring in the same patient, and suggest that lineage plasticity may occur in mature lymphoid neoplasms.