Abstract
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.
Gerrie:AstraZeneca, Beigene, Janssen, Lilly: Research Funding; AstraZeneca, Beigene, Janssen, Lily, Celgene: Honoraria. Inghirami:Daiichi Sankyo: Consultancy. Holte:Incyte: Consultancy; Pierre Fabre: Consultancy; SERB: Consultancy. Feldman:Zeno Pharmaceuticals: Patents & Royalties; Seattle Genetics: Research Funding. Savage:Bristol Myers Squibb: Consultancy, Research Funding; AbbVie: Consultancy; Seagen: Consultancy, Honoraria, Research Funding; Regeneron: Other: DSMC. Steidl:Bayer: Consultancy; EISAI: Consultancy; Seattle Genetics: Consultancy; Epizyme: Research Funding; Bristol Myers Squibb: Research Funding; AbbVie: Consultancy; Trillium Therapeutics Inc: Research Funding. Lenz:Novartis: Honoraria, Research Funding; Karyopharm: Honoraria; Genmab: Honoraria; Constellation: Honoraria; Lilly: Honoraria; Immagene: Honoraria; Incyte: Honoraria; Hexal/Sandoz: Honoraria; Genase: Honoraria; BeiGene: Honoraria; BMS: Honoraria; Amgen: Honoraria; AbbVie: Honoraria; ADC Therapeutics: Honoraria; Verastem: Research Funding; Roche: Honoraria, Research Funding; MSD: Honoraria; NanoString: Honoraria; PentixaPharm: Honoraria; Miltenyi Biotech: Honoraria; Pierre Fabre: Honoraria; Sobi: Honoraria, Speakers Bureau; Acerta: Research Funding; Roche, Gilead, BMS, Novartis, AstraZeneca, Abbvie, Incyte, Genmab, Constellation, ADC Therapeutics, Miltenyi, PentixaPharm, Sobi, Immagene, Genase, Hexal-Sandoz, Lilly, Beigene, MSD, Pierre Fabre: Consultancy; ELVESCA: Current equity holder in private company; Roche, Gilead, BMS, Novartis, AstraZeneca, Abbvie, Incyte, Genmab, Constellation, ADC Therapeutics, Miltenyi, PentixaPharm, Sobi, Immagene, Genase, Hexal-Sandoz, Lilly, Beigene, MSD, Pierre Fabre: Membership on an entity's Board of Directors or advisory committees; AbbVie, BeiGene, Sobi, Roche, Gilead, BMS: Other: Travel; MorphoSys: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; AQUINOX: Research Funding; AstraZeneca: Honoraria, Research Funding; Bayer: Honoraria, Research Funding; AGIOS: Research Funding. Scott:AstraZenenca: Consultancy; Janssen: Consultancy; Roche: Research Funding; Incyte: Consultancy; Abbvie: Consultancy.