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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.

Follicular lymphoma (FL) accounts for approximately 20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been comprehensively described. In this study, we analyzed whole genome sequencing data from 423 patients to compare the protein coding and non-coding mutation landscapes of untransformed FL, transformed FL and de novo DLBCL. This revealed two genetically distinct subgroups of FL which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, biological, and clinical characteristics. We implemented a machine-learning-derived classification approach to stratify FL patients into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution and we highlight the potential for this classification to predict HT from genetic features present at diagnosis.

Prognostic values of MHC expression

IHC antibodies and cell line information

Supplementary Methods, Figures and Legends

Genetic backgrounds of MHC deficient cases

Genetic analysis in the MHC-I/II double-positive cases

We performed a genomic, transcriptomic, and immunophenotypic study of 347 patients with diffuse large B-cell lymphoma (DLBCL) to uncover the molecular basis underlying acquired deficiency of MHC expression. Low MHC-II expression defines tumors originating from the centroblast-rich dark zone of the germinal center (GC) that was associated with inferior prognosis. MHC-II–deficient tumors were characterized by somatically acquired gene mutations reducing MHC-II expression and a lower amount of tumor-infiltrating lymphocytes. In particular, we demonstrated a strong enrichment of EZH2 mutations in both MHC-I– and MHC-II–negative primary lymphomas, and observed reduced MHC expression and T-cell infiltrates in murine lymphoma models expressing mutant Ezh2Y641. Of clinical relevance, EZH2 inhibitors significantly restored MHC expression in EZH2-mutated human DLBCL cell lines. Hence, our findings suggest a tumor progression model of acquired immune escape in GC-derived lymphomas and pave the way for development of complementary therapeutic approaches combining immunotherapy with epigenetic reprogramming.Significance:We demonstrate how MHC-deficient lymphoid tumors evolve in a cell-of-origin–specific context. Specifically, EZH2 mutations were identified as a genetic mechanism underlying acquired MHC deficiency. The paradigmatic restoration of MHC expression by EZH2 inhibitors provides the rationale for synergistic therapies combining immunotherapies with epigenetic reprogramming to enhance tumor recognition and elimination.See related commentary by Velcheti et al., p. 472.This article is highlighted in the In This Issue feature, p. 453

EZH2 mutation data

Gene expression analyses of clinical samples

MHC expression status in the cohort

Supplementary Table 1 from Acquired TNFRSF14 Mutations in Follicular Lymphoma Are Associated with Worse Prognosis

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.

Objectives Intravascular large B-cell lymphomas (IVLBCLs) are rare extranodal LBCLs in which relapse is relatively frequent. We sought to further characterize potential immune escape mechanisms in IVLBCLs that newer therapies can exploit. Methods A series of 33 IVLBCLs were evaluated for programmed cell death ligand 1 (PD-L1) and PD-L2 expression by immunohistochemistry (IHC), chromosomal alterations (CAs) in the PDL1/PDL2 locus by fluorescence in situ hybridization, and loss of major histocompatibility complex (MHC) class I and II expression by IHC. Results Cases were subclassified as classical (n = 22) or hemophagocytic syndrome (HPS)–associated (n = 11) variants. A total of 12 cases (39%; n = 12/31) expressed PD-L1 and/or PD-L2. CAs were seen in 7 cases (7/29 [24%]) and included gains, amplifications, and rearrangements. CAs in classical variant cases (24%; n = 5/21) included gains (n =1), gains with concurrent rearrangements (n = 2), and amplifications (n = 2). The 2 HPS-associated variant cases with CAs (25%; n = 2/8) both showed amplification, including 1 case with a concurrent rearrangement. A majority of cases with CAs (71%; n = 5/7) were PD-L1/PD-L2 IHC positive. Among PD-L1/PD-L2 IHC–positive cases, 45% harbored a CA. Loss of MHC class I and/or class II was seen in 27% (n = 9/33) of cases. Conclusions Altogether, our data show that 65% (n = 20/31) of IVLBCLs may exploit immune evasion strategies through PD-L1/PD-L2 expression or downregulation of MHC proteins.

High programmed cell death 1 ligand 1 (PD-L1) protein expression and copy number alterations (CNAs) of the corresponding genomic locus 9p24.1 in Hodgkin- and Reed-Sternberg cells (HRSC) have been shown to be associated with favourable response to anti-PD-1 checkpoint inhibition in relapsed/refractory (r/r) classical Hodgkin lymphoma (cHL). In the present study, we investigated baseline 9p24.1 status as well as PD-L1 and major histocompatibility complex (MHC) class I and II protein expression in 82 biopsies from patients with early stage unfavourable cHL treated with anti-PD-1-based first-line treatment in the German Hodgkin Study Group (GHSG) NIVAHL trial (ClinicalTrials.gov Identifier: NCT03004833). All evaluated specimens showed 9p24.1 CNA in HRSC to some extent, but with high intratumoral heterogeneity and an overall smaller range of alterations than reported in advanced-stage or r/r cHL. All but two cases (97%) showed PD-L1 expression by the tumour cells in variable amounts. While MHC-I was rarely expressed in >50% of HRSC, MHC-II expression in >50% of HRSC was found more frequently. No obvious impact of 9p24.1 CNA or PD-L1 and MHC-I/II expression on early response to the highly effective anti-PD-1-based NIVAHL first-line treatment was observed. Further studies evaluating an expanded panel of potential biomarkers are needed to optimally stratify anti-PD-1 first-line cHL treatment.

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry and digital GEP to assign cell-of-origin (COO) and the so-called "double-hit signature" (DHITsig) - a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2-year overall survival of 57%, 89% and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared to DZsig-expressing HGBCL-DH-BCL2, and were associated with favorable outcomes comparable DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit–like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.

Primary mediastinal large B-cell lymphoma (PMBL) represents a clinically and pathologically distinct subtype of large B-cell lymphomas. Furthermore, molecular studies, including global gene expression profiling, have provided evidence that PMBL is more closely related to classical Hodgkin lymphoma (cHL). Although targeted sequencing studies have revealed a number of mutations involved in PMBL pathogenesis, a comprehensive description of disease-associated genetic alterations and perturbed pathways is still lacking. Here, we performed whole-exome sequencing of 95 PMBL tumors to inform on oncogenic driver genes and recurrent copy number alterations. The integration of somatic gene mutations with gene expression signatures provides further insights into genotype–phenotype interrelation in PMBL. We identified highly recurrent oncogenic mutations in the Janus kinase-signal transducer and activator of transcription and nuclear factor κB pathways, and provide additional evidence of the importance of immune evasion in PMBL (CIITA, CD58, B2M, CD274, and PDCD1LG2). Our analyses highlight the interferon response factor (IRF) pathway as a putative novel hallmark with frequent alterations in multiple pathway members (IRF2BP2, IRF4, and IRF8). In addition, our integrative analysis illustrates the importance of JAK1, RELB, and EP300 mutations driving oncogenic signaling. The identified driver genes were significantly more frequently mutated in PMBL compared with diffuse large B-cell lymphoma, whereas only a limited number of genes were significantly different between PMBL and cHL, emphasizing the close relation between these entities. Our study, performed on a large cohort of PMBL, highlights the importance of distinctive genetic alterations for disease taxonomy with relevance for diagnostic evaluation and therapeutic decision-making.

Gray-zone lymphoma (GZL) with features intermediate between classic Hodgkin lymphoma (cHL) and large B-cell lymphoma (LBCL) was introduced as a provisional entity into the World Health Organization classification in 2008. However, as diagnostic criteria are imprecise, reliable identification of GZL cases remains challenging. Here, we describe the histopathologic features of 139 GZL cases from a retrospective Lymphoma Study Association (LYSA) study with the goal to improve classification accuracy. Inclusion criteria were based on literature review and an expert consensus opinion of the LYSA hematopathologist panel. We observed 86 cases with a morphology more closely related to cHL, but with an LBCL immunophenotype based on strong and homogenous B-cell marker expression (CD20 and/or CD79a, OCT2, BOB1, PAX5) on all tumor cells (cHL-like GZL). Fifty-three cases were morphologically more closely related to LBCL but harbored a cHL immunophenotype (LBCL-like GZL). Importantly, we observed a continuous morphologic and immunophenotypic spectrum within these 2 GZL categories. The majority of cases presented genetic immune escape features with CD274/PDCD1LG2 and/or CIITA structural variants by fluorescence in situ hybridization. Patients without mediastinal involvement at diagnosis (17%) were older than those with mediastinal tumors (median: 56 vs. 39 y). Cases associated with Epstein-Barr virus (24%) presented with similar patient characteristics and outcome as Epstein-Barr virus negative cases. In summary, we provide refined diagnostic criteria that contribute to a more precise pathologic and clinical characterization of GZL within a broad spectrum from cHL-like to LBCL-like disease.

Significance Our study provides detailed functional and spatial characteristics of immune cells in the LR-CHL microenvironment at single-cell resolution. We describe detailed T cell subset definitions and importantly identified a unique CD4+PD-1+CXCL13+CXCR5− TFH-like subset that surrounds HRS cells, appears in close proximity to CXCR5+ B cells, and is associated with poor clinical outcome. We also uncovered unique PD-1/PD-L1 axis biology in LR-CHL, namely a negative correlation between PD-L1 genetic alterations on HRS cells and PD-1 protein expression in the tumor microenvironment. Importantly, our findings contribute to a deeper understanding of cellular cross-talk in LR-CHL, which may aid in the development of novel biomarkers and targeted treatment strategies., Lymphocyte-rich classic Hodgkin lymphoma (LR-CHL) is a rare subtype of Hodgkin lymphoma. Recent technical advances have allowed for the characterization of specific cross-talk mechanisms between malignant Hodgkin Reed-Sternberg (HRS) cells and different normal immune cells in the tumor microenvironment (TME) of CHL. However, the TME of LR-CHL has not yet been characterized at single-cell resolution. Here, using single-cell RNA sequencing (scRNA-seq), we examined the immune cell profile of 8 cell suspension samples of LR-CHL in comparison to 20 samples of the mixed cellularity (MC, 9 cases) and nodular sclerosis (NS, 11 cases) subtypes of CHL, as well as 5 reactive lymph node controls. We also performed multicolor immunofluorescence (MC-IF) on tissue microarrays from the same patients and an independent validation cohort of 31 pretreatment LR-CHL samples. ScRNA-seq analysis identified a unique CD4+ helper T cell subset in LR-CHL characterized by high expression of Chemokine C-X-C motif ligand 13 (CXCL13) and PD-1. PD-1+CXCL13+ T cells were significantly enriched in LR-CHL compared to other CHL subtypes, and spatial analyses revealed that in 46% of the LR-CHL cases these cells formed rosettes surrounding HRS cells. MC-IF analysis revealed CXCR5+ normal B cells in close proximity to CXCL13+ T cells at significantly higher levels in LR-CHL. Moreover, the abundance of PD-1+CXCL13+ T cells in the TME was significantly associated with shorter progression-free survival in LR-CHL (P = 0.032). Taken together, our findings strongly suggest the pathogenic importance of the CXCL13/CXCR5 axis and PD-1+CXCL13+ T cells as a treatment target in LR-CHL.

The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, were analyzed as a discovery cohort, followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) exhibited a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (n = 18) had a significantly distinct pattern that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV− GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas.

When the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture.

Genomic rearrangements in the MYC locus occur in ∼12% of lymphomas with diffuse large B-cell lymphoma (DLBCL) morphology and are associated with inferior outcome. Previous studies exploring MYC rearrangements have primarily used fluorescence in situ hybridization (FISH) assays to characterize break-apart status but have rarely examined breakpoint location, and in some cases have not examined partner identity. We performed targeted sequencing of MYC, BCL2, BCL6, and the immunoglobulin (IG) loci in 112 tumors with DLBCL morphology harboring MYC rearrangement. We characterized the location of the MYC rearrangement at base pair resolution and identified the partner in 88 cases. We observed a cluster of breakpoints upstream of the MYC coding region and in intron 1 (the “genic cluster”). Genic cluster rearrangements were enriched for translocations involving IGH (80%), whereas nongenic rearrangements occurred mostly downstream of the MYC gene with a variety of partners, including IGL and IGK. Other recurrent partners included BCL6, ZCCHC7, and RFTN1, which has not previously been described as a MYC partner. We compared 2 commercially available FISH break-apart assays for the MYC locus and observed discordant results in 32% of cases examined, including some with MYC-IGL and MYC-IGK rearrangements. In cases of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement (HGBL-DH), so-called “double-hit” lymphomas, the majority of MYC rearrangements had non-IG partners (65%), with breakpoints outside the genic cluster (72%). In patients with de novo HGBL-DH of DLBCL morphology, MYC-IG rearrangements showed a trend toward inferior time to progression and overall survival compared with MYC–non-IG rearrangements. Our data reveal clinically relevant architecture of MYC rearrangements in lymphomas with DLBCL morphology.

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management.

Introduction: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH), colloquially referred to as double- or triple-hit lymphoma, is associated with poor outcomes prompting many centers to use dose-intensive immunochemotherapy. The benefit of treatment intensification in patients with HGBL-DH/TH with diffuse large B-cell lymphoma (DLBCL) morphology, who would otherwise receive standard-of-care rituximab, cyclophosphamide, vincristine and prednisone (R-CHOP), remains unclear because suitably powered randomized clinical trials have not been performed, and earlier studies included patients with high-grade morphology. Furthermore, selection bias due to the restriction of fluorescence in situ hybridization (FISH) testing to diagnose HGBL-DH/TH in patients with high-risk clinical presentation or aggressive tumor morphology confounds historical comparators. Since 2015, de novo DLBCL biopsies in British Columbia (BC) have undergone routine FISH testing in clinical practice. Concurrently, provincial guidelines were introduced recommending treatment with dose-adjusted etoposide, doxorubicin, vincristine, cyclophosphamide, prednisone and rituximab (DA-EPOCH-R) for appropriately fit patients aged 75 years (y) or younger with HGBL-DH/TH harboring BCL2 rearrangements with DLBCL morphology (HGBL-DH/TH-BCL2-DLBCL). A population-based analysis was conducted to assess the impact of the introduction of DA-EPOCH-R on outcomes in HGBL-DH/TH-BCL2-DLBCL. Methods: Outcomes of HGBL-DH/TH-BCL2-DLBCL patients diagnosed between 2015 to 2020 using clinical FISH performed on de novo DLBCL biopsies (DA-EPOCH-R era) were compared to patients with HGBL-DH/TH-BCL2-DLBCL identified from a historic province-wide cohort of de novo DLBCL diagnosed between 2005-2010 in BC that underwent universal FISH in a research setting (R-CHOP era). Patients with the rarer entity of HGBL-DH/TH harboring MYC and BCL6 rearrangements only (HGBL-DH-BCL6) were excluded and were not part of the original DA-EPOCH-R guideline. Multivariable Cox proportional hazards regression models were used to predict the independent effect of treatment era after controlling for the International Prognostic Index (IPI). Results: 99 patients with HGBL-DH/TH-BCL2-DLBCL were identified through routine clinical FISH in the DA-EPOCH-R era. Of 1172 de novo DLBCL patients in the historic R-CHOP era, 824 had adequate diagnostic material for evaluation by FISH, 52 of which were HGBL-DH/TH-BCL2-DLBCL. The analysis was restricted to patients aged 75y or younger, yielding 71 and 38 patients in the DA-EPOCH-R and R-CHOP eras, respectively. 7/38 (18%) biopsies in the R-CHOP era had undergone clinical FISH testing at diagnosis with results known to the treating physician. Median (interquartile range) follow-up in living patients was 2.8y (2.0-4.4y) in the DA-EPOCH-R era and 12.2y (11.2-13.4y) in the R-CHOP era. 49/71 (69%) patients received DA-EPOCH-R in the DA-EPOCH-R era, whereas 32/38 patients (84%) received R-CHOP in the R-CHOP era. Both eras had comparable baseline clinical characteristics with no significant difference in IPI risk groups (Table 1). The DA-EPOCH-R era was associated with superior 2-year time to progression (TTP, 73.9% vs 47.4%, p=0.016) and overall survival (OS, 77.7% vs 50.0%, p=0.022, Figure 1). After adjusting for IPI risk groups (low 0-2, high 3-5), the DA-EPOCH-R era was independently associated with superior TTP (hazard ratio (HR) 0.41, 95% confidence interval (CI) 0.21-0.77, p=0.005) and OS (HR 0.39, 95% CI 0.21-0.76, p=0.005). After controlling for individual IPI factors, treatment era remained predictive of TTP (HR 0.38, 95% CI 0.19- 0.76, p=0.006) and OS (HR 0.39, 95% CI 0.19-0.79, p=0.008). Conclusions: Introduction of a provincial, population-based recommendation to use DA-EPOCH-R for appropriately fit patients aged 75y or younger is associated with improved real-world outcomes of HGBL-DH/TH-BCL2-DLBCL. The similarity between TTP and OS within each era suggests the high failure rate of conventional salvage therapy irrespective of frontline treatment, prompting further investigation of novel second-line therapies in this poor-prognosis population. Targeted capture sequencing to identify MYC translocation partners is underway, and the influence of the MYC partner on outcomes in both eras will be presented. Figure 1 Figure 1. Disclosures Sehn: Novartis: Consultancy; Genmab: Consultancy; Debiopharm: Consultancy. Slack: Seagen: Consultancy, Honoraria. Craig: Bayer: Consultancy. Villa: Janssen: Honoraria; Gilead: Honoraria; AstraZeneca: Honoraria; AbbVie: Honoraria; Seattle Genetics: Honoraria; Celgene: Honoraria; Lundbeck: Honoraria; Roche: Honoraria; NanoString Technologies: Honoraria. Gerrie: Sandoz: Honoraria; Roche: Research Funding; Astrazeneca: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Janssen: Honoraria, Research Funding. Freeman: Teva: Research Funding; Roche: Research Funding; Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria; Celgene: Honoraria; Sanofi: Honoraria, Speakers Bureau; Incyte: Honoraria; Abbvie: Honoraria; Seattle Genetics: Honoraria; Bristol Myers Squibb: Honoraria, Speakers Bureau. Savage: Seattle Genetics: Consultancy, Honoraria; Roche: Research Funding; Astra-Zeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Other: Institutional clinical trial funding; BMS: Consultancy, Honoraria, Other: Institutional clinical trial funding; Servier: Consultancy, Honoraria; Takeda: Other: Institutional clinical trial funding; Beigene: Other: Institutional clinical trial funding; Genentech: Research Funding. Steidl: Bayer: Consultancy; Trillium Therapeutics: Research Funding; Curis Inc.: Consultancy; AbbVie: Consultancy; Epizyme: Research Funding; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding. Scott: Celgene: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; 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. ; Rich/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Incyte: Consultancy; Abbvie: Consultancy; AstraZeneca: Consultancy.

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..

Introduction: Follicular lymphoma (FL) is an indolent disease that undergoes histological transformation (HT) to aggressive diffuse large B-cell lymphoma (DLBCL) in 8-15% of patients. FLs frequently share genetic features with DLBCL, especially those of the germinal center B-cell-like (GCB) cell-of-origin (COO) and the EZB/C3 genetic subgroup, and approximately 80% of transformed FL (tFL) are classified as GCB. Our current understanding of the genetics of FL and tFL is based on a variety of studies, most of which have sequenced tumors in small case numbers or using targeted approaches such that the potential role of non-coding mutations and aberrant somatic hypermutation (aSHM) in predicting HT have not previously been fully explored. Methods: Whole genome sequencing (WGS) data from 212 FL (including 24 from patients that subsequently underwent HT) and 241 de novo DLBCL were analyzed. Simple somatic mutations (SSMs) were called using an ensemble of somatic variant callers, while structural variants (SVs) were called with Manta and copy number variants (CNVs) with Battenberg and GISTIC. Fluorescence in situ hybridization with break-apart probes (BA-FISH) was used to identify MYC, BCL2, and BCL6 translocations, and with IGH /BCL2 dual-fusion probes (DF-FISH) for a subset of FLs. To compare the genetics of FL and DLBCL, 83 significantly mutated genes (SMGs) were identified with dNdS, MutSigCV, HotMaps, and OncoDriveFML, and non-silent mutations were tabulated for their presence in each genome. For 38 hypermutated regions, we used a region-specific threshold to binarize the data to aSHM/no aSHM. Recurrent missense mutations in FOXO1, MYD88L265P, CREBBP lysine acetyltransferase (KAT) domain, EZH2Y646, MEF2B, and STAT6 were tabulated separately from other mutations in these genes. Using only the FL tumors from patients with no evidence of subsequent transformation and all available de novo DLBCLs, we trained a random forest classifier to separate these two entities using this set of 129 features, including MYC and BCL6 translocations. To validate this classifier, we fit a linear model to the number of FL votes from each discovery case, utilizing the 65 features (including 19 aSHM features) that were adequately sequenced in a validation cohort of 127 tFL. Statistical tests were corrected for multiple comparisons where necessary. Results: This large cohort of FL and DLBCL genomes has enabled the curation of an extensive list of novel and established FL driver genes and the identification of distinguishing genetic features among SMGs and CNVs. Loci that are significantly enriched for mutations in FL vs. DLBCL include the CREBBP KAT domain (OR 11.5, P < 0.0001), RRAGC (OR 9.61, P < 0.001), and ATP6V1B2 (OR 11.17, P < 0.001). Deletions of ARID1A (OR 4.74, P < 0.1), PTEN (OR 3.65, P < 0.01), and TNFRSF14 (OR 3.31, P < 0.01) were among the CNVs significantly enriched in FL. Out of 156 FLs, 24 (15%) were negative for BCL2 translocations by BA-FISH, but 4 (17%) of these had BCL2 translocations detected from WGS data. All 4 of these cryptic events were confirmed using IGH /BCL2 DF-FISH. Using a threshold of 0.7, the linear model separated discovery FL cases into a more DLBCL-like subgroup, termed dFL (n = 107), and a genetically homogeneous subgroup enriched for the FL-associated features, which we describe as constrained FL (cFL, n = 105). This separation is supported by more mutations in dFL vs cFL across several aSHM loci, including the transcription start sites for BCL6, BCL7A, DTX1, and ZFP36L1 (Figure 1), consistent with reduced exposure to the germinal center reaction in cFL. Within the targeted capture validation cohort of tFL, 30 (24%) tumors were classified as cFL and 97 (76%) as dFL. The tFL cohort was significantly depleted for mutations in the CREBBP KAT domain (OR 0.59, P < 0.05), and were significantly less frequently classified as cFL (OR 0.30, P < 0.0001) compared to the discovery FLs. Conclusions: The distinction between cFL and dFL is strongly driven by CREBBP KAT domain mutations and different rates of aSHM genome wide. Given the known early clonal nature of CREBBP mutations in FL and its role in regulating germinal center cycling, we speculate that CREBBP KAT mutations may limit the exposure of FL to the dark zone, reducing the opportunity for aSHM and creating an evolutionary constraint that may limit the opportunity for HT. This classification may serve as a useful biomarker to identify FLs at higher risk of HT. Figure 1 Figure 1. Disclosures Coyle: Allakos, Inc.: Consultancy. Grande: Sage Bionetworks: Current Employment. Slack: Seagen: Consultancy, Honoraria. Steidl: Curis Inc.: Consultancy; Trillium Therapeutics: Research Funding; Epizyme: Research Funding; Bayer: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Bristol-Myers Squibb: Research Funding. Scott: Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; NanoString Technologies: Patents & Royalties: Patent describing measuring the proliferation signature in MCL using gene expression profiling.; Incyte: Consultancy; Rich/Genentech: Research Funding; Celgene: Consultancy; 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. . Morin: Epizyme: Patents & Royalties; Foundation for Burkitt Lymphoma Research: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

The clinical significance of MYC and BCL2 genetic alterations in diffuse large B-cell lymphoma (DLBCL), apart from translocations, has not been comprehensively investigated using high-resolution genetic assays. In this study, we profiled MYC and BCL2 genetic alterations using next-generation sequencing and high-resolution SNP array in 347 de novo DLBCL cases treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) at the British Columbia Cancer Agency. Cell-of-origin (COO) subtype was determined by Lymph2Cx digital gene expression profiling. We showed that the incidence of MYC/BCL2 genetic alterations and their clinical significance were largely dependent on COO subtypes. It is noteworthy that the presence of BCL2 gain/amplification is significantly associated with poor outcome in activated B-cell-like and BCL2 translocation with poor outcome in germinal center B-cell subtypes, respectively. Both have prognostic significance independent of MYC/BCL2 dual expression and the International Prognostic Index (IPI). Furthermore, the combination of BCL2 genetic alterations with IPI identifies markedly worse prognostic groups within individual COO subtypes. Thus, high-resolution genomic assays identify extremely poor prognostic groups within each COO subtype on the basis of BCL2 genetic status in this large, uniformly R-CHOP-treated population-based cohort of DLBCL. These results suggest COO subtype-specific biomarkers based on BCL2 genetic alterations can be used to risk-stratify patients with DLBCL treated with immunochemotherapy.

Introduction: Classic Hodgkin lymphoma (CHL) features a unique crosstalk between malignant cells and different types of normal immune cells in the tumor-microenvironment (TME). On the basis of histomorphologic and immunophenotypic features of the malignant Hodgkin and Reed-Sternberg (HRS) cells and infiltrating immune cells, four histological subtypes of CHL are recognized: Nodular sclerosing (NS), Mixed cellularity, Lymphocyte-rich (LR) and Lymphocyte-depleted CHL. Recently, our group described the high abundance of various types of immunosuppressive CD4+ T cells including LAG3+ and/or CTLA4+ cells in the TME of CHL using single cell RNA sequencing (scRNAseq). However, the TME of LR-CHL has not been well characterized due to the rarity of the disease. In this study, we aimed at characterizing the immune cell profile of LR-CHL at single cell resolution. METHODS: We performed scRNAseq on cell suspensions collected from lymph nodes of 28 primary CHL patients, including 11 NS, 9 MC and 8 LR samples, with 5 reactive lymph nodes (RLN) serving as normal controls. We merged the expression data from all cells (CHL and RLN) and performed batch correction and normalization. We also performed single- and multi-color immunohistochemistry (IHC) on tissue microarray (TMA) slides from the same patients. In addition, an independent validation cohort of 31 pre-treatment LR-CHL samples assembled on a TMA, were also evaluated by IHC. Results: A total of 23 phenotypic cell clusters were identified using unsupervised clustering (PhenoGraph). We assigned each cluster to a cell type based on the expression of genes described in published transcriptome data of sorted immune cells and known canonical markers. While most immune cell phenotypes were present in all pathological subtypes, we observed a lower abundance of regulatory T cells (Tregs) in LR-CHL in comparison to the other CHL subtypes. Conversely, we found that B cells were enriched in LR-CHL when compared to the other subtypes and specifically, all four naïve B-cell clusters were quantitatively dominated by cells derived from the LR-CHL samples. T follicular helper (TFH) cells support antibody response and differentiation of B cells. Our data show the preferential enrichment of TFH in LR-CHL as compared to other CHL subtypes, but TFH cells were still less frequent compared to RLN. Of note, Chemokine C-X-C motif ligand 13 (CXCL13) was identified as the most up-regulated gene in LR compared to RLN. CXCL13, which is a ligand of C-X-C motif receptor 5 (CXCR5) is well known as a B-cell attractant via the CXCR5-CXCL13 axis. Analyzing co-expression patterns on the single cell level revealed that the majority of CXCL13+ T cells co-expressed PD-1 and ICOS, which is known as a universal TFH marker, but co-expression of CXCR5, another common TFH marker, was variable. Notably, classical TFH cells co-expressing CXCR5 and PD-1 were significantly enriched in RLN, whereas PD-1+ CXCL13+ CXCR5- CD4+ T cells were significantly enriched in LR-CHL. These co-expression patterns were validated using flow cytometry. Moreover, the expression of CXCR5 on naïve B cells in the TME was increased in LR-CHL compared to the other CHL subtypes We next sought to understand the spatial relationship between CXCL13+ T cells and malignant HRS cells. IHC of all cases revealed that CXCL13+ T cells were significantly enriched in the LR-CHL TME compared to other subtypes of CHL, and 46% of the LR-CHL cases showed CXCL13+ T cell rosettes closely surrounding HRS cells. Since PD-1+ T cell rosettes are known as a specific feature of LR-CHL, we confirmed co-expression of PD-1 in the rosetting cells by IHC in these cases. Conclusions: Our results reveal a unique TME composition in LR-CHL. LR-CHL seems to be distinctly characterized among the CHL subtypes by enrichment of CXCR5+ naïve B cells and CD4+ CXCL13+ PD-1+ T cells, indicating the importance of the CXCR5-CXCL13 axis in the pathogenesis of LR-CHL. Figure Disclosures Savage: BeiGene: Other: Steering Committee; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy. Scott:Janssen: Consultancy, Research Funding; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy. Steidl:AbbVie: Consultancy; Roche: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding.