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PURPOSEAbout a third of relapsed or refractory classic Hodgkin lymphoma (r/r CHL) patients succumb to their disease after high-dose chemotherapy followed by autologous stem cell transplantation (HDC/ASCT). Here, we aimed to describe spatially resolved tumor microenvironment (TME) ecosystems to establish novel biomarkers associated with treatment failure in r/r CHL.METHODSWe performed imaging mass cytometry (IMC) on 169 paired primary diagnostic and relapse biopsies using a marker panel specific for CHL biology. For each cell type in the TME, we calculated a ’spatial score’ measuring the distance of nearest neighbor cells to the malignant Hodgkin Reed Sternberg cells within close interaction range. ‘Spatial scores’ were used as features in prognostic model development for post-ASCT outcomes.RESULTSHighly multiplexed IMC data revealed shared TME patterns in paired diagnostic and early relapse/refractory CHL samples, whereas TME patterns were more divergent in pairs of diagnostic and late relapse samples. Integrated analysis of IMC and single cell RNA sequencing data identified unique architecture defined by CXCR5+HRS cells and their strong spatial relationship with CXCL13+ macrophages in the TME. We developed a prognostic assay (‘RHL4S’) using four spatially resolved parameters, CXCR5+ HRS cells, PD1+CD4+ T cells, tumor-associated macrophages, and CXCR5+ B cells, which effectively separated patients into high-risk vs low-risk groups with significantly different post-ASCT outcomes. The RHL4S assay was validated in an independent r/r CHL cohort using a multicolor immunofluorescence assay.CONCLUSIONSWe identified the interaction of CXCR5+ HRS cells with ligand-expressing CXCL13+ macrophages as a prominent crosstalk axis in relapsed CHL. Harnessing this TME biology, we developed a novel prognostic model applicable to r/r CHL biopsies, RHL4S, opening new avenues for spatial biomarker development.

Diffuse large B-cell lymphoma (DLBCL) is cured in over 60% of patients, but outcomes are poor for patients with relapsed or refractory disease (rrDLBCL). Here, we performed whole genome/exome sequencing (WGS/WES) on tumors from 73 serially-biopsied patients with rrDLBCL. Based on the observation that outcomes to salvage therapy/autologous stem cell transplantation are related to time-to-relapse, we stratified patients into groups according to relapse timing to explore the relationship to genetic divergence and sensitivity to salvage immunochemotherapy. The degree of mutational divergence increased with time between biopsies, yet tumor pairs were mostly concordant for cell-of-origin, oncogene rearrangement status and genetics-based subgroup. In patients with highly divergent tumors, several genes acquired exclusive mutations independently in each tumor, which, along with concordance of genetics-based subgroups, suggests that the earliest mutations in a shared precursor cell constrain tumor evolution. These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease.

Mutations affecting enhancer chromatin regulatorsCREBBPandKMT2Dare highly co-occurrent in germinal center (GC)-derived lymphomas and other tumors, even though regulating similar pathways. Herein, we report that combined haploinsufficiency ofCrebbpandKmt2d(C+K) indeed accelerated lymphomagenesis. C+K haploinsufficiency induced GC hyperplasia by altering cell fate decisions, skewing B cells away from memory and plasma cell differentiation. C+K deficiency particularly impaired enhancer activation for immune synapse genes involved in exiting the GC reaction. This effect was especially severe at super-enhancers for immunoregulatory and differentiation genes. Mechanistically, CREBBP and KMT2D formed a complex, were highly co-localized on chromatin, and were required for each-other’s stable recruitment to enhancers. Notably, C+K lymphomas in mice and humans manifested significantly reduced CD8+T-cell abundance. Hence, deficiency of C+K cooperatively induced an immune evasive phenotype due at least in part to failure to activate key immune synapse super-enhancers, associated with altered immune cell fate decisions.SIGNIFICANCEAlthough CREBBP and KMT2D have similar enhancer regulatory functions, they are paradoxically co-mutated in lymphomas. We show that their combined loss causes specific disruption of super-enhancers driving immune synapse genes. Importantly, this leads to reduction of CD8 cells in lymphomas, linking super-enhancer function to immune surveillance, with implications for immunotherapy resistance.

Follicular lymphoma (FL) is the most common indolent form of non-Hodgkin lymphoma. Histological transformation of FL to a more aggressive form of lymphoma occurs with a linear incidence of 2-3% per year and is associated with poor outcome. Divergent clonal evolution and an altered tumour microenvironment (TME) have both been implicated in the transformation process. However, the phenotypic consequences of this evolution and its implication in reshaping the TME remain unknown. To address this knowledge gap we performed single cell whole genome (scWGS) and single cell whole transcriptome sequencing (scWTS) of paired pre/post transformation samples of 11 FL patients. We further performed scWTS analysis of additional 11 FL samples from patients that had not undergone transformation within 7 years. Our comprehensive single cell analysis revealed the evolutionary dynamics of transformation at unprecedented resolution. Computational integration of scWGS and scWTS allowed us to identify gene programs upregulated and positively selected during evolution. Furthermore, our scWTS analysis revealed a shifting TME landscape, with an exhausted CD8 T cell signature emerging during transformation. Using multi-color immunofluorescence we transferred these findings to a novel TME based biomarker of transformation, subsequently validated in 2 independent cohorts of pretreatment FL samples. Taken together, our results provide a comprehensive view of the combined genomic and phenotypic evolution of malignant cells during transformation, and the shifting cross-talk between malignant cells and the TME.Graphical abstract

Burkitt lymphoma (BL) accounts for the majority of pediatric non-Hodgkin lymphomas (NHL) and is relatively rare but significantly more lethal when diagnosed in adults. The global incidence is highest in Sub-Saharan Africa, where Epstein-Barr virus (EBV) positivity is observed in 95% of all tumors. Both pediatric (pBL) and adult (aBL) cases are known to share some driver mutations, for example MYC translocations, which are seen in > 90% of cases. Sequencing efforts have identified many common somatic alterations that cooperate with MYC in lymphomagenesis with approximately 30 significantly mutated genes (SMG) reported thus far. Recent analyses revealed non-coding mutation patterns in pBL that were attributed to aberrant somatic hypermutation (aSHM). We sought to identify genomic and molecular features that may explain clinical disparities within and between aBL and pBL in an effort to delineate BL subtypes that may allow for the stratification of patients with shared pathobiology. Through comprehensive sequencing of BL genomes, we found additional SMGs, including more genetic features that associate with tumor EBV status, and established three new genetic subgroups that span pBL and aBL. Direct comparisons between pBL and aBL revealed only marginal differences and the mutational profiles were consistently better explained by EBV status. Using an unsupervised clustering approach to identify subgroupings within BL and diffuse large B-cell lymphoma (DLBCL), we have defined three genetic subgroups that predominantly comprise BL tumors. Akin to the recently defined DLBCL subgroups, each BL subgroup is characterized by combinations of common driver mutations and non-coding mutations caused by aSHM. Two of these subgroups and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among the aBL and pBL cohorts. These findings highlight not only a shared pathogenesis between aBL and pBL, but also establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiological studies, and diagnostic and therapeutic strategies.

BackgroundCancers often overexpress multiple clinically relevant oncogenes. However, it is not known if multiple oncogenes within a cancer combine uniquely in specific cellular sub-populations to influence clinical outcome. We studied this phenomenon using the prognostically relevant oncogenes MYC, BCL2 and BCL6 in Diffuse Large B-Cell Lymphoma (DLBCL).MethodsQuantitative multispectral imaging simultaneously measured oncogene co-expression at single-cell resolution in reactive lymphoid tissue (n=12) and four independent cohorts (n=409) of DLBCL. Mathematically derived co-expression phenotypes were evaluated in DLBCLs with immunohistochemistry (n=316) and eight DLBCL cohorts with gene expression data (n=4186). Bulk and single-cell RNA sequencing was performed on patient-derived B-cells with induced co-expression of MYC, BCL2 and BCL6.ResultsUnlike in non-malignant lymphoid tissue where the co-expression of MYC, BCL2 and BCL6 in a B-cell is limited, DLBCLs show multiple permutations of oncogenic co-expression in malignant B-cells. The percentage of cells with a unique combination MYC+BCL2+BCL6-(M+2+6-) consistently predicts survival in contrast to that of other combinations (including M+2+6+). An estimated percentage of M+2+6-cells can be derived from any quantitative measurement of the component individual oncogenes, and correlates with survival in immunohistochemistry and gene expression datasets. Comparative transcriptomic analysis of DLBCLs and transformed patient-derived B-cells identifies cyclin D2 (CCND2) as a potential BCL6-repressed regulator of proliferation in the M+2+6-population.ConclusionsUnique patterns of oncogene co-expression at single-cell resolution affect clinical outcomes in DLBCL. Similar analyses evaluating oncogenic combinations at the cellular level may impact diagnostics and target discovery in other cancers.

Binary classification using gene expression data is commonly used to stratify cancers into molecular subgroups that may have distinct prognoses and therapeutic options. A limitation of many such methods is the requirement for comparable training and testing data sets. Here, we describe and demonstrate aself-training implementation ofprobabilityratio-based classificationpredictionscore (PRPS-ST) that facilitates the porting of existing classification models to other gene expression data sets. We demonstrate its robustness through application to two binary classification problems in diffuse large B-cell lymphoma using a diverse variety of gene expression data types and normalization methods.

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established and the features known to contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes alongside previously published exome cohorts. To confirm our findings, we re-sequenced the genes identified in the exome cohort in 212 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association ofTP53andNOTCH1mutations and further nominate two additional genes,EWSR1andMEF2B, whose mutation respectively associated with poor and good outcome. Our sequencing revealed novel recurrent mutations including a unique missense hot spot inMEF2Band a pattern of non-coding mutations surrounding a single exon of theHNRNPH1gene. We sequenced the whole genomes of 34 MCLs to confirm the focal nature ofHNRNPH1mutations. Using RNA-seq data from 110 of these cases, we identified a functional role for recurrent non-codingHNRNPH1mutations in disrupting an auto-regulatory feedback mechanism. Overall, we identified three novel MCL-related genes with roles in RNA trafficking or splicing, namelyDAZAP1, EWSR1, andHNRNPH1. Taken together, these data strongly implicate a role for aberrant regulation of splicing in MCL pathobiology.Key pointsRNA-binding proteins with roles in regulating alternative splicing,DAZAP1, EWSR1, HNRNPH1, are frequently mutated in MCLThe majority of recurrent somaticHNRNPH1mutations are intronic and HNRNPH1 exhibits self-regulation through alternative splicing

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Many known driver mutations are over-represented in one of its two molecular subgroups, knowledge of which has aided in the development of therapeutics that target these features. The heterogeneity of DLBCL determined through prior genomic analysis suggests an incomplete understanding of its molecular aetiology, with a limited diversity of genetic events having thus far been attributed to the activated B-cell (ABC) subgroup. Through an integrative genomic analysis we uncovered genes and non-coding loci that are commonly mutated in DLBCL including putative regulatory sequences. We implicate recurrent mutations in the 3’UTR of NFKBIZ as a novel mechanism of oncogene deregulation and found small amplifications associated with over-expression of FC-γ receptor genes. These results inform on mechanisms of NF-κB pathway activation in ABC DLBCL and may reveal a high-risk population of patients that might not benefit from standard therapeutics.