Mutations In Diffuse Large B-cell Lymphoma Research Articles

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin’s lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6− tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

Crebbp Kat Domain Missense Mutations Alter Its Dynamic Loading Onto Chromatin and Create a Transcription Factor Sink

The CREBBP gene encodes a lysine acetyltransferase (KAT) and is one of the most frequently mutated genes in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). However, the spectrum of mutations is significantly different between these two diseases, with significantly higher fractions of nonsense/frameshift mutations in DLBCL and comparatively higher fractions of lysine acetyltransferase (KAT) domain point mutations (KAT-PM) in FL. Among KAT domain mutations, multiple hotspots can be observed with R1446 (14%), Y1482 (3%) and Y1503 (7%) being the most frequently mutated residues. We have previously shown that CREBBP R1446C mutations lead to a more profound loss of histone acetylation and gene expression compared to biallelic knock-out (KO) of the CREBBP highlighting a potential dominant-negative function of R1446C via an unknown mechanism. We have also performed biochemical and structural analysis of KAT domain mutations and observed that R1446C mutations result in a partial loss of function (pLOF), whereas Y1482N and Y1503C mutations are catalytically dead. Here, we report the molecular characterization of CRISPR-engineered isogenic cell lines (RL, HT, 293T) bearing biallelic KO, R1446C, Y1482N or Y1503C mutations, or WT CREBBP, using CUT&RUN analysis of CREBBP and EP300 loading, histone marks (H3K27Ac, H3K4me1, H3K4me2, H3K27me3), and ATAC and RNA sequencing for chromatin accessibility and gene expression, respectively. Western blot analysis showed significant reduction of H3K27Ac and H3K18Ac in all point-mutants with no alteration of CREEBP or its paralog EP300 expression. Principal component analysis (PCA) of H3K27Ac and gene expression data revealed that KO cells were most similar to WT, and all KAT-PM cells clustered separately to WT and KO cells. Notably, R1446C (partial LOF) cells reproducibly clustered separately from Y1482N and Y1503C (catalytically dead), and differential analysis identified both shared and R1446C- or Y1482N/Y1503-specific losses of H3K27Ac in all cell lines, suggesting that the degree of loss in catalytic activity rendered by different KAT domain point mutations may be associated with distinct molecular phenotypes. In order to investigate the mechanism by which KAT-PMs drive more severe molecular changes compared to biallelic KO, we investigate the redundant role of EP300 using CUT&RUN analysis of EP300 loading. CREBBP-bound active enhancers (H3K4me1+ H3K27Ac+) showed a significant increase in EP300 loading in cells with CREBBP KO compared to WT. However, we observed a slight decrease in EP300 loading within KAT-PM cells, indicating that the presence of mutant CREBBP protein prevents the redundant activity of EP300. We speculated that this may be due to mutant CREBBP residing on chromatin in an inactive state and preventing loading of EP300. We therefore analyzed CREBBP loading over CREBBP-bound active enhancers in WT cells that had significant reductions of H3K27Ac in KAT-PMs. Surprisingly, we observed a significant reduction in CREBBP loading over these regions, with a greater loss of CREBBP loading in catalytically dead mutants (Y1482N/Y1503C) compared to the partial LOF mutant (R1446C). We therefore characterized patterns of CREBBP loading during normal germinal center B-cell development by analyzing purified tonsillar naïve B-cells, centroblasts and centrocytes. We observed that CREBBP is dynamically loaded onto chromatin during the centroblast-to-centrocyte transition, indicating that loss of chromatin-bound CREBBP in KAT-PMs may be due to a failure of mutant CREBBP to load onto these sites. We next performed transcription factor (TF) motif enrichment analysis and identified a significant enrichment of key TFs such as BACH2 and IRF4 in regions with failed CREBBP loaded and EP300 compensation, indicating that CREBBP may bind these transcription factors and act as a sink that prevents redundant EP300 activity. We validated this using tetracycline-inducible over-expression of IRF4 in KAT-PM cells, which increased EP300 loading and H3K27Ac over IRF4 binding sites. Collectively, our results indicate that CREBBP KAT-PMs prevent the redundant activity of EP300 in a manner proportionally to their relative loss of catalytic activity, by preventing the dynamic loading of CREBBP onto chromatin and acting as a transcription factor sink.

Diffuse large B-cell lymphoma and red cell autoimmunity: clinical role and pathogenesis

Diffuse large B-cell lymphoma (DLBCL) is the most common form of B-cell non-Hodgkin lymphoma (B-NHL) with significant morbidity and mortality despite advancements in treatment. Lymphoma and autoimmune disease both result from breakdowns in normal cell regulatory pathways, and epidemiological studies have confirmed both that B-NHL is more likely to develop in the setting of autoimmune diseases and vice versa. Red cell immunity, as evidenced by direct antiglobulin test (DAT) positivity, has been linked to DLBCL and more recently the pathogenic causes of this association have begun to be better understood using molecular techniques. This project aimed to explore the relationship between red cell autoimmunity and DLBCL. DAT positivity was more common in DLBCL as compared to healthy controls (20.4% vs 3.7%, p=0.0005). Univariate analysis found a non-significant trend towards poorer overall survival in the DAT positive (DAT+) compared to the DAT negative (DAT-) groups (p=0.087). High throughput sequencing was used to compare mutations in DLBCL from DAT+ and DAT- patients. The most frequently mutated genes in 15 patient samples were KMT2D (n=13), MYOM2 (n=9), EP300 (n=8), SPEN (n=7), and ADAMTSL3 (n=7), which were mutated in both DAT+ and DAT- groups. BIRC3 (n=3), FOXO1 (n=3) and CARD11 (n=2) were found to be mutated only in samples from the DAT+ group. These gene mutations may be involved in disease development and progression, and potentially represent targets for future therapy. The immunoglobulin genotype IGHV4-34 is seen more frequently in DLBCL clones than in normal B cells and has intrinsic autoreactivity to self-antigens on red cells, which is largely mediated by two motifs within the first framework region (FR1); Q6W7 and A24V25Y.26 These motifs form a hydrophobic patch which determines red cell antigen binding and are frequently mutated away from self-reactivity in normal B cells. If this does not occur this may provide constant B cell receptor signalling which encourages lymphoma development, a theory known as antigen driven lymphomagenesis. As with previous studies, IGHV4-34 was over-represented (15.6%) in our DLBCL cohort. Furthermore, of 6 IGHV4-34-expressing DLBCL samples five had unmutated hydrophobic patch mutations providing further evidence for antigen-driven lymphomagenesis. Mutation analysis of these five samples demonstrated high frequency of mutations in several genes, including CREBBP and NCOR2. Further research could explore if mutations in CREBBP and NCOR2 work in conjunction with the preserved QW and AVY motifs to promote lymphomagenesis in IGHV4-34-expressing B cells, and if so, could guide future targeted therapy.

P1235: GENOTYPING ON CIRCULATING TUMOR DNA IMPROVES MUTATION DETECTION RATE IN HIGH-RISK DIFFUSE LARGE B-CELL LYMPHOMA

Background: Diffuse large B cell lymphoma (DLBCL) is one of the most common lymphomas with heterogeneous clinical outcome. The circulating tumor DNA (ctDNA) represents a novel non-invasive source of tumor genetic material that overcomes anatomical biases of tissue biopsy, and thus has been widely used in aggressive lymphomas. Aims: In this study, we collected a total of 170 samples from 104 DLBCL patients with different disease status, including 62 with paired ctDNA and tissue biopsy genomic DNA (gDNA) samples, to determine the clinical relevance of ctDNA genotyping as well as its concordance with tissue biopsy gDNA genotyping. Methods: A targeted sequencing gene panel including all exons and critical introns of 112 genes that are recurrently mutated in DLBCL was used for this study (Burning Rock Biotech Co. Ltd, Guangzhou, China). The samples were divided into 4 groups according to the disease status at enrollment: ① treatment-sensitive (TS), representing samples from 39 treatment-naïve DLBCLs who responded to frontline treatment; ② primary refractory (Pri-R), representing samples from 10 treatment naïve DLBCLs who failed to achieve partial remission (PR) at the interim evaluation or complete remission (CR) at the end of frontline treatment; ③ early-relapse/refractory (ERR), representing samples collected at the time of relapse from 45 patients who relapsed within 12 months after diagnosis or refractory to prior treatment; ④ late-relapse (LR), representing samples collected at the time of relapse from 13 patients who relapsed more than 12 months after diagnosis. Results: With a mean sequencing depth of 16140× (range: 8377×−23356×) in ctDNA and 1868× (range: 1009×−2881×) in gDNA, variants were identified in 88.2% (90/102) ctDNAs and profiles were similar to that of gDNA genotyping. The mean VAF of ctDNA mutations showed a good correlation with elevated lactate dehydrogenase (LDH, P=0.0008) and β2-microglobulin (β2-MG, P=0.039), as well as the metabolism tumor volume (MTV, R 0.38; P=0.0024) and the total lesion glycolysis (TLG, R 0.40; P=0.0012) assessed by PET/CT. Besides, patients with a mean VAF of ≥1.5% (determined by X-tile) had significantly shorter PFS (P=0.0004) and OS (P=0.0024). In the 62 patients who had paired ctDNA and gDNA samples, we found ctDNA genotyping outperformed gDNA genotyping in ERR DLBCLs. The proportion of ctDNA-specific variants in ERR DLBCLs was significantly higher than that of the other three groups (TS: 7.3%, 29/398; Pri-R: 10.3%, 4/39; ERR: 25.9%, 225/870; LR: 3.2%, 4/126; P<0.001). We further investigated the profile of ctDNA-specific mutations in ERR DLBCLs and found that a considerable amount of unfavorable gene mutations, including 33.3% (4/12) of TP53 mutations, 66% (4/6) of PRDM1 mutations, and 62.5% (5/8) of EP300 mutations.On the other hand, the detection rate of genetic subtypes was 51.6% (32/62) and 50% (31/62) in ctDNA and gDNA in the 62 patients with paired samples.When the two sample types were combined together, the detection rate of genetic subtypes could reach 58.1% (36/62). Interestingly, it was found that all but one (6/7) ctDNA-specific subtype belonged to the MCD subtype. In fact, 35.3% (6/17) MCD DLBCLs were only identified by ctDNA but not by gDNA, suggesting the addition of ctDNA genotyping substantially improved the detection rate of MCD subtype (figure 1). Image:Summary/Conclusion: Our study highlights the clinical application of ctDNA, in particular in patients with high-risk and early relapsed or refractory diseases, where ctDNA analysis represents a complementary approach to tissue biopsy gDNA genotyping.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell–mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

Conformational transitions in BTG1 antiproliferative protein and their modulation by disease mutants

B cell translocation gene 1 (BTG1) protein belongs to the BTG/transducer of ERBB2 (TOB) family of antiproliferative proteins whose members regulate various key cellular processes such as cell cycle progression, apoptosis, and differentiation. Somatic missense mutations in BTG1 are found in ∼70% of a particularly malignant and disseminated subtype of diffuse large B cell lymphoma (DLBCL). Antiproliferative activity of BTG1 has been linked to its ability to associate with transcriptional cofactors and various enzymes. However, molecular mechanisms underlying these functional interactions and how the disease-linked mutations in BTG1 affect these mechanisms are currently unknown. To start filling these knowledge gaps, here, using atomistic molecular dynamics (MD) simulations, we explored structural, dynamic, and kinetic characteristics of BTG1 protein, and studied how various DLBCL mutations affect these characteristics. We focused on the protein region formed by α2 and α4 helices, as this interface has been reported not only to serve as a binding hotspot for several cellular partners but also to harbor sites for the majority of known DLBCL mutations. Markov state modeling analysis of extensive MD simulations revealed that the α2-α4 interface in the wild-type (WT) BTG1 undergoes conformational transitions between closed and open metastable states. Importantly, we show that some of the mutations in this region that are observed in DLBCL, such as Q36H, F40C, Q45P, E50K (in α2), and A83T and A84E (in α4), either overstabilize one of these two metastable states or give rise to new conformations in which these helices are distorted (i.e., kinked or unfolded). Based on these results, we conclude that the rapid interconversion between the closed and open conformations of the α2-α4 interface is an essential component of the BTG1 functional dynamics that can prime the protein for functional associations with its binding partners. Disruption of the native dynamic equilibrium by DLBCL mutants leads to the ensemble of conformations in BTG1 that are unlikely structurally and/or kinetically to enable productive functional interactions with the binding proteins.

Influence of TP53 Mutation on Survival of Diffuse Large B-Cell Lymphoma in the CAR T-Cell Era.

Refractory/relapsed diffuse large B-cell lymphoma (DLBCL) is associated with poor outcome. The clinical behavior and genetic landscape of DLBCL is heterogeneous and still not fully understood. TP53 mutations in DLBCL have been identified as markers of poor prognosis and are often associated with therapeutic resistance. Chimeric antigen receptor T-cell therapy is an innovative therapeutic concept and represents a game-changing therapeutic option by supporting the patient's own immune system to kill the tumor cells. We investigated the impact of TP53 mutations on the overall survival of refractory/relapsed DLBCL patients treated with comparable numbers of therapy lines. The minimum number of therapy lines was 2 (median 4), including either anti-CD19 CAR T-cell therapy or conventional salvage therapy. A total of 170 patients with DLBCL and high-grade B-cell lymphoma with MYC, BCL2, and/or BCL6 rearrangements (DHL/THL), diagnosed and treated in our hospital between 2000 and 2021, were included. Twenty-nine of them received CAR T-cell therapy. TP53 mutations were found in 10/29 (35%) and 31/141 (22%) of patients in the CAR T-cell and conventional groups, respectively. Among the 141 patients not treated with CAR T cells, TP53 mutation was an independent prognostic factor for overall survival (OS) (median 12 months with TP53 vs. not reached without TP53 mutation, p < 0.005), but in the CAR T cell treated group, this significance could not be shown (median OS 30 vs. 120 months, p = 0.263). The findings from this monocentric retrospective study indicate that TP53 mutation status does not seem to affect outcomes in DLBCL patients treated with CAR T-cell therapy. Detailed evaluation in large cohorts is warranted.

TET Deficiency Is Associated with Accumulation of G-Quadruplex and R-Loop Structures during Oncogenesis

The three members of TET family of Fe(II) and alpha-ketoglutarate-dependent dioxygenases mediate DNA demethylation by sequentially oxidizing 5-methylcytosine (5mC) to 5-hydroxymethyl- (5hmC), 5-formyl- (5fC) and 5-carboxyl-cytosine (5caC). TET enzymes are required for normal development, and loss of TET function due to mutations, metabolic perturbations and hypoxia, among other mechanisms, occurs frequently in many hematological malignancies and solid tumors. Recent studies have identified mutations in TET proteins (TET2, most commonly) and metabolic enzymes which regulate TET catalytic activity in a large cohort of patients with Diffuse Large B-cell Lymphoma (DLBCL). However, the clinical significance of these mutations in DLBCL and the molecular mechanisms through which TET proteins suppress development of malignancies in general, are not fully-understood.To investigate the role of TET loss-of-function in the pathogenesis of DLBCL, we generated mice with B-cell-specific deletion of TET2 and TET3, the major TET homologs expressed in mature B cells. TET deficiency in B cells perturbed mature B cell homeostasis resulting in spontaneous development of Germinal Center-derived B cell lymphomas. Moreover, B cells with TET deficiency demonstrated increased genomic instability, a feature previously associated with TET loss-of-function in other hematopoietic lineages. Transcriptional profiling of TET-deficient expanded B cells revealed altered expression of genes and proteins involved in modulating the levels of secondary DNA structures, G-quadruplexes and DNA:RNA hybrids (R-loops) which have been linked to genomic instability and transcriptional perturbations in many different cancers. Using previously described methods and newer approaches, we observed a substantial increase in the levels of G-quadruplex and R-loop structures in TET-deficient B cells compared with control B cells. The increase in G-quadruplex and R-loop structures was evident in naïve, activated and GC B cells following acute TET deletion as well as in TET-deficient myeloid cells and T cells. Genome-wide mapping studies and high-throughput genome-wide translocation sequencing (HTGTS) showed a strong correlation of increased G-quadruplex and R-loop structures with increased DNA DSBs in switch regions of immunoglobulin heavy chain locus in TET-deficient B cells. Deletion of the DNA methyltransferase DNMT1 in TET-deficient B cells prevented the expansion of germinal center B cells, diminished the accumulation of G-quadruplexes and R-loops, and caused a notable delay in lymphoma development, consistent with the opposing functions of DNMT and TET enzymes in DNA methylation and demethylation. CRISPR-mediated depletion of nucleases and helicases that regulate G-quadruplexes and R-loops decreased the viability of TET-deficient B cells. Our studies suggest a molecular mechanism by which TET loss-of-function might predispose to development of B cell-derived and other malignancies, and highlight novel therapeutic avenues that could be further explored. DisclosuresRao: Cambridge Epigenetix: Membership on an entity's Board of Directors or advisory committees.

Aging-Associated Epigenetic Reprogramming Alters the Germinal Center Reaction and Targets Pathways Related to Lymphomagenesis

Aging is associated with suboptimal germinal center (GC) responses and inferior outcomes of GC-derived lymphomas such as diffuse large B-cell lymphoma (DLBCL). At the molecular level, aging is characterized by epigenetic alterations of DNA CpG methylation and chromatin architecture that ultimately affect cellular functions. The B-cell/T-cell immune synapse during the transitional stages of the GC is governed by a series of epigenetic switches. Frequent mutations in DLBCL directly impact epigenetic regulators and signaling pathways in the immune synapse. Here, we investigated the impact of aging-associated epigenetic alterations in the functionality of the GC response in parallel with biological characteristics of DLBCL in the elderly.We conducted an integrative characterization of epigenomic, transcriptomic and phenotypic changes of B and T cells during the GC reaction in younger (3-4 months) vs. older (21-23 months) mice by immunophenotyping (flow cytometry), DNA methylation sequencing of sorted sub-populations, single cell (SC) RNA-sequencing (coding and BCR/TCR) and SC-multiomics (integration of RNA-sequencing and ATAC-sequencing). We also performed DNA methylation sequencing, RNA-sequencing and NGS in a cohort of 266 DLBCL including 51 pts >75 y.o.In agreement with reported phenotypes in elderly humans (Collier D. et al., Nature, 2021), the GC reaction in older mice was characterized by a significative reduction in IgG3+ (p=0.0001) and increased in IgM+ (p=0.009) B cells (FAS+ CD38-); whereas the non-GC B cell compartment (FAS- CD38+) displayed an increase in age-associated B cells (ABCs, p=0.0001) and reduction in follicular B-cells (p=0.004). Furthermore, older mice displayed an expansion of a FAS+CD38+ population of B cells (p=0.0001). Regarding T cells, we observed a global reduction in CD4+ (p=0.01) but not in CD8+ cells; however, older mice showed an expansion of PD1+/CD4+ and PD1+/CD8+ T cells (p=0.0004 and p=0.0003, respectively). Furthermore, older mice displayed increased TFH (p=0.0001), Tregs (p=0001) and ICOS+ Treg (p=0001) populations. SC-RNA-seq of B and T cells validated these alterations and identified transcriptionally-defined sub-populations including expansion of clonal GZMK CD8+ TOX+ T cells (TAA cells) and activation of “cytokine production” in T cells (FDR=3.01e-27), both phenotypes associated with “inflammaging” (Mogilenko et al., Immunity, 2020). Epigenetic changes in older B-cells showed aberrant hypermethylation of gene promoters and hypomethylation of intergenic regions including enhancers, resulting in changes of chromatin architecture and gene expression. In GC B-cells but not naïve B-cells (NBC), genes whose promoter was aberrantly hypermethylated in older mice were enriched for targets of SUZ12 (FDR= 5.1e -12), EZH2 (FDR= 5.1e -12) and JARID2 (FDR= 6.8e -10), key components of the PRC2 complex. Older mice B-cells also displayed decreased chromatin accessibility in genes functionally enriched for “semaphorin-plexin signaling pathway” (FDR=5.3e -03) which regulates TFH/B-cell immune synapse as well as decreased expression of linker histone H1 isoforms B-E in GCB cells (q=0.0006; q=0.0003; q=0.008, q=4.64E-10, respectively).Notably, the age-associated reprogramming observed in the germinal center reaction globally targets pro-tumorigenic pathways that are comparably altered in elderly DLBCL. We observed that older pts (>75 y.o.) vs. younger pts (55-65 y.o.) had increased hypermethylation of gene promoters and hypomethylation of intergenic regions with deregulation of epigenetic modifiers (including PCR2 members) and immune synapse genes (including BCR signaling). There were no differences in the prevalence of specific mutations between these two populations. However, DLBCL in elderly are more frequently of the ABC subtype (~60%, p<0.05) and presented with an inflammatory tumor microenvironment (41% vs 27% in younger DLBCL, p<0.001, corrected by COO) that is characterized by increased infiltration of macrophages (p=0.001), Tregs (p=0.001) and CD8+ PD1 highcells (p=0.001), phenocopying changes observed in the germinal center of older mice.Thus, age-associated epigenetic reprogramming results in perturbation of pathways regulating the B-cell/T-cell immune synapse during the normal germinal center reaction and may underlie some of the biological characteristics observed in elderly DLBCL patients. DisclosuresLara-Garcia: Johnson and Johnson: Current holder of individual stocks in a privately-held company; Bristol Myers Squibb: Divested equity in a private or publicly-traded company in the past 24 months; Moderna: Divested equity in a private or publicly-traded company in the past 24 months; Pfizer: Divested equity in a private or publicly-traded company in the past 24 months; Regeneron: Divested equity in a private or publicly-traded company in the past 24 months; Merck: Divested equity in a private or publicly-traded company in the past 24 months; Fortress Biotech: Divested equity in a private or publicly-traded company in the past 24 months. Cerchietti: Celgene: Research Funding; Bristol Myers Squibb: Research Funding.

Prognostic and therapeutic value of somatic mutations in diffuse large B-cell lymphoma: A systematic review.

Diffuse large B-cell lymphoma (DLBCL), the most common type of Non-Hodgkin lymphoma (NHL), is a highly heterogeneous and aggressive disease. Regardless of this heterogeneity, all patients receive the same first-line therapy, which fails in 30-40 % of patients, who are either refractory or relapse after remission. With the aim of stratifying patients to improve treatment outcome, different clinical and genetic biomarkers have been studied. The present systematic review aimed to identify somatic mutations that could serve as prognosis biomarkers or as therapeutic target mutations in DLBCL. Regarding their role as prognostic markers, mutations in CD58 and TP53 seem the most promising predictors of poor outcome although the combination of different alterations and other prognostic factors could be a more powerful strategy. On the other hand, different approaches regarding targeted therapy have been proposed. Therefore, mutational analysis could help guide treatment choice in DLBCL yet further studies and clinical trials are needed.

Integrative Analysis of 1001 Diffuse Large B Cell Lymphoma Identifies Novel Oncogenic Roles for Rhoa

Abstract Diffuse large B cell lymphoma (DLBCL) is the most common hematologic malignancy and is characterized by a striking degree of clinical and molecular heterogeneity. Gene expression profiling has long been used to define subtypes and understand the heterogeneity within DLBCL. Microarray-based gene expression studies have identified cell of origin subgroups (Alizadeh et al., 2000), as well as gene expression signatures derived from host inflammatory response (Monti et al., 2005), stromal tissues (Lenz et al., 2008) as well as other biological processes. However, the connections between these gene expression signatures and genetic alterations is largely unknown. In this study, we sought to define connections between gene expression signatures and genetic alterations to better understand the biology of DLBCL. We enrolled a total of 1001 DLBCL patients and comprehensively defined the landscape of genetic mutations, copy number alterations and expression through whole-exome and transcriptome sequencing. We identified 150 genetic drivers of DLBCL including many novel, clinically relevant genes (e.g. SPEN, SETD1B and KLHL14). We began the gene expression analysis with a comprehensive universe of nearly 9500 annotated gene sets from widely used gene set databases (Kegg, Reactome, MSigDB), several lymphoma-specific gene signature sources (Lenz et al. 2008, Monti et al. 2005, and Shaffer et al.) and other published sources. From this large universe, we identified 1228 gene sets that comprised genes with high intra gene set correlation. The vast majority of these 1228 gene sets also exhibited high inter-gene set correlation indicating a high degree of redundancy between these gene sets. We developed an approach to singular value decomposition to define 31 exemplar gene sets that essentially replicated the patterns of the 1228 described gene sets. Our results recapitulated a number of described patterns underlying DLBCL gene expression including distinct gene sets reflecting cell of origin, host response, BCR and Oxidative Phosphorylation (Monti et al., 2005), as well as the “Stromal 1” and “Stromal 2” signatures (Lenz et al., 2008) signatures. There was no association between the expression of stromal signatures and overall mutational burden (p=0.4). Our data identified the genetic underpinnings of each of these distinct gene sets that provide a better understanding of the underlying biology affected by different genetic mutations. Interestingly, genetic alterations in RHOA were strongly associated with the proliferation gene signature and poor survival in DLBCL. We sought to better define the role of RHOA mutations in DLBCL which are mostly unknown. We knocked out the expression of RHOA in vitro through the CRISPR-Cas9 approach and found that RHOA deletion was lethal in both ABC DLBCL (LY3, TMD8, HBL1) and GCB DLBCLs (Pfeiffer and SUDHL4), results were validated with shRNAs. Conversely, cells overexpressing R5Q mutant proliferated 30% faster than vector control cells and more than 10% faster than wildtype overexpressing cells, directly implicating these mutations and RHOA overexpression in the growth of DLBCL cells. We further investigated the effects of RHOA knockout in vivo using two complementary mouse models. We bred Rhoa conditional knockout mice with Mb1-Cre transgenic mice, in which Cre is restricted to the B cell lineage as well as AID-Cre transgenic mice in which Cre is restricted to germinal center (GC) B cells. While B cells represented the vast majority of cells in the lymphoid organs of Rhoa wildtype mice, we found a dramatic reduction in B cell population and a resulting increase in T cell percentages in Rhoa deleted mice (p Collectively, our data define the genetic basis for observed changes in gene expression in DLBCL. Our experimental data define RHOA as an oncogene with roles in B cell development, germinal center maintenance, and DLCBL growth. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding; Merck: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen Cilag: Consultancy, Research Funding. Flowers: Abbvie: Consultancy, Research Funding; Infinity: Research Funding; Clinical Care Options: Research Funding; National Cancer Institute: Research Funding; Spectrum: Consultancy; Onyx: Research Funding; OptumRx: Consultancy; Prime Oncology: Research Funding; Janssen Pharmaceutical: Research Funding; Acerta: Research Funding; Gilead: Consultancy; Seattle Genetics: Consultancy; Eastern Cooperative Oncology Group: Research Funding; Research to Practice: Research Funding; Burroughs Welcome Fund: Research Funding; Celgene: Consultancy, Research Funding; V Foundation: Research Funding; Bayer: Consultancy; Educational Concepts: Research Funding; Pharmacyclics LLC, an AbbVie Company: Research Funding; Genentech/Roche: Consultancy, Research Funding; Millennium/Takeda: Research Funding; National Institutes Of Health: Research Funding; TG Therapeutics: Research Funding. Gribben: TG Therapeutics: Honoraria; Kite: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Pharmacyclics: Honoraria; Abbvie: Honoraria; Acerta: Honoraria; Karyopharm: Honoraria; Genentech/Roche: Honoraria. Hsi: Cellerant Therapeutics: Research Funding; Abbvie: Research Funding; Seattle Genetics: Consultancy, Honoraria, Speakers Bureau; Eli Lilly and Co.: Research Funding. Evens: Merck: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Millennium: Consultancy; AbbVie: Consultancy; Affimed: Consultancy; Pharmacyclics: Consultancy; Celgene: Consultancy; Kite Pharma: Consultancy. Reddy: Abbvie: Consultancy; BMS: Consultancy; Celgene: Consultancy; Gilead: Speakers Bureau. Gordon: Janssen: Other: Data Monitoring Committee.

Combined Genotype Analysis of Tumor and Cell-Free DNA By Ultra-Deep Targeted Sequencing: Correlation with PET-Scan Imaging in a Prospective Cohort of Diffuse Large B-Cell Lymphoma Patients

The emerging concept of liquid biopsy in diffuse large B-cell lymphoma (DLBCL) is based on the release of Cell-free DNA (cfDNA) into the blood by tumor cells undergoing apoptosis or necrosis that can be used for the identification of mutations (Bohers et al., Haematologica 2015 ; Rossi et al., Blood 2017). cfDNA theoretically can give therefore information regarding potentially actionable mutations, clonal evolution, and genetic mechanisms of resistance. To determine the clinical relevance of cfDNA, we assessed prospectively by Ultra-deep targeted next generation sequencing (NGS) the basal DLBCL genetic pattern, its modification upon treatment using plasma cfDNA analysis and its correlation with PET scan imaging (clinical.gov number: NCT02339805).MethodsThirty consecutive untreated DLBCL or primary mediastinal B-cell lymphoma (PMBL) were enrolled and followed after rituximab-cyclophosphamide-doxorubicin-vincristine-prednisone (R-CHOP) or R-CHOP-like chemotherapies. Mutational status of a 34-genes lymphopanel (CIITA, B2M, TNFRSF14, CD58, NOTCH1, NOTCH2, MFHAS1, XPO1, MYC, CDKN2A/B, FOXO1, TP53, GNA13, BCL2, TNFAIP3, MYD88, PIM1, CARD11, IRF4, PRDM1, EZH2, KMT2D, EP300, MEF2B, CREBBP, BRAF, CD79A/B, ITPKB, TCF3, ID3, SOCS1 and STAT6) was assessed in both tumor (genomic DNA, gDNA) and cfDNA at the time of diagnosis using AmpliSeq®/PGM® technologies (Dubois et al., Clinical Cancer Research 2016) . Longitudinal analysis of plasma samples collected at mid-treatment (C4) and at the end of treatment or at relapse/progression was performed and correlated with PET scan characteristics obtained at baseline (Metabolic tumor volume, MTV), mid and end-treatment (Delta SUV and Deauville score). To increase the NGS sensitivity and for cost-effectiveness reasons, only mutations detected at baseline were monitored during the follow-up. Variant analysis was performed using an in-house generated bioinformatics pipeline.ResultsPatient clinical features were as follow: IPI: 0-1, n =8; IPI: 2-3, n=14; IPI: 4-5, n =8; median age was 64 y; bone marrow involvement: 0/25 patients; elevated LDH: 12/30; Fifteen patients were sub-classified as GCB, 9 as ABC/non GCB and 3 as PMBL. cfDNA was available for the 30 patients at the time of diagnosis. Mean plasma concentration was 74 ng/mL (14.6 - 433) and sequencing was carried out with a mean depth of 3750x. cfDNA mutations were identified in 20 patients (67%) and were consistent with the pattern usually observed in the different subtypes of DLBCL. gDNA mutations from tissue biopsies (11 frozen/14 FFPE) were found in 22/25 (88%) cases. In 3/22 cases for whom gDNA analysis failed, an informative mutational pattern was finally successfully obtained in matched cfDNA. Variant allelic frequency (VAF) was lower in cfDNA (mean 15%) compared to tumor DNA (mean 33%). Finally, among the 22 tumor/plasma matched samples, 8 patients had no detectable tumoral cfDNA, whereas 4 patients displayed additional mutations in plasma compared to tumor. At the time of diagnosis, a high [cfDNA] correlated with the presence of tumoral circulating DNA and cfDNA mutations were observed in all patients with elevated LDH. MTV correlated with the highest concentrations of cfDNA and VAF rate. Moreover, 3 / 4 patients presenting additional mutations in cfDNA, as compared to gDNA, had a high MTV (> 2000cm3), suggesting that cfDNA mutations reflect more accurately tumor heterogeneity than gDNA analysis. After R-CHOP/ R-CHOP-like regimens, a rapid clearance of cfDNA mutations was observed in 13/17 cases. Conversely, among the 4/17 patients whose basal DLBCL mutations did not disappear at mid-treatment, 3 were in partial response according to Deauville score, including 2 with a delta SUVmax < 70%. One patient in partial remission had progressive disease 6 months after the end of the treatment and still harboured tumoral cfDNA, as shown by a persistent detectable TP53 mutation.ConclusionOur study demonstrates that cfDNA genotyping of DLBCL is an accurate genotyping tool and represents a real-time and non-invasive approach to tracking treatment-resistant clones. It highlights the major interest of the liquid biopsy in the context of bulky tumors where cfDNA is more representative than gDNA by capturing the entire tumoral mutation profile. cfDNA analysis represents therefore a complementary approach to TEP scan imaging at baseline and during follow-up for the management of DLBCL. DisclosuresStamatoullas:Takeda: Consultancy; Celgene Corporation: Honoraria. Tilly:Karyopharm: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immunogen: Honoraria; Gilead: Honoraria; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Honoraria. Jardin:Roche: Honoraria; celgen: Honoraria; Janssen: Honoraria.

SGK1 mutations in DLBCL generate hyperstable protein neoisoforms that promote AKT independence

Serum and glucocorticoid-regulated kinase 1 (SGK1) is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL). However, little is known about its function or the consequence of its mutation. The frequent finding of truncating mutations has led to the widespread assumption that these represent loss-of-function variants and, accordingly, that SGK1 must act as a tumor suppressor. In this study, instead, the most common SGK1 mutations led to production of aberrantly spliced messenger RNA neoisoforms in which translation is initiated from downstream methionines. The resulting N-terminal truncated protein isoforms showed increased expression related to the exclusion of an N-terminal degradation domain. However, they retained a functional kinase domain, the overexpression of which rendered cells resistant to AKT inhibition, in part because of increased phosphorylation of GSK3B. These findings challenge the prevailing assumption that SGK1 is a tumor-suppressor gene in DLBCL and provide the impetus to explore further the pharmacological inhibition of SGK1 as a therapeutic strategy for DLBCL.

The Tumor Associated Antigen PRAME Exhibits Dualistic Functions That Are Targetable in Diffuse Large B-Cell Lymphoma

Background: With the goal of translating biological discovery into clinical actionability, deciphering crosstalk in the cellular ecosystem of the tumor microenvironment (TME) has emerged as a research focus. Although comparatively little is known about the immune biology of diffuse large B-cell lymphoma (DLBCL), as reflected in clonal selection of specific somatic gene mutations in response to immune system pressure and the specific composition of the TME, PRAME has emerged as a prominent member of the cancer germline antigen/ tumor associated antigen (TAA) family of proteins. It is expressed in various types of cancers, but generally not in normal tissues, apart from male germinal cells, and triggers autologous T-cell mediated immune responses. PRAME is highlighted as a new cancer therapeutic target of T-cell or antibody-based immunotherapies with promising anti-tumor responses in early phase clinical trials and pre-clinical models for several types of cancers. In the context of developing new immunotherapies, targeting TAAs that are presented by major histocompatibility complexes on tumor cells is a promising therapeutic strategy for patients that experience treatment failure. Material and methods: We performed integrative genomic analysis of whole-transcriptome RNAseq, targeted genomic sequencing, and high-resolution copy number analysis in 347 de novo DLBCL tumors from patients uniformly treated with R-CHOP. Findings were correlated with pathological and clinical parameters, as well as TME composition. Using DLBCL-derived cell lines (7 EZH2 mutated and 5 wt) and CRISPR-Cas9 genome editing, we performed in vitro functional studies to characterize cell-intrinsic effects of PRAME knockout. Moreover, we studied EZH2 inhibition in a murine model of Ezh2 mutant lymphoma with a focus on mechanistic links between EZH2 activity and PRAME expression, as well as TME composition (cell-extrinsic effects). Results: We discovered recurrent, and highly focal deletions of 22q11.22 including the PRAME gene, which were associated with poor treatment outcome, independent of pathological and clinical risk factors. To explore PRAME-deletion-associated phenotypes and interaction with the tumor microenvironment (TME), we analyzed corresponding RNAseq, immunohistochemistry (IHC), and flow-cytometry data from our DLBCL cohort and utilized enzyme-linked immunospot (ELISPOT) assays using patient-derived peripheral blood mononuclear cells. PRAME deletions contributed to an immunologically "cold" TME, representing a somatically acquired mechanism to evade anti-tumor T-cell response (cell-extrinsic effect). Using PRAME knock out by CRISPR-Cas9 in vitro, TRAIL-mediated apoptotic signaling was impaired. In addition, PRAME down-modulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. Using proximity ligation assays and co-IP, we demonstrated that PRAME directly interacted with EZH2 as a negative regulator, establishing a link between PRAME deletions and EZH2 mutations with anti-apoptotic signaling in DLBCL (cell-intrinsic effect). An in vivo murine model of Ezh2 mutant lymphoma showed decreased T-cell infiltration in the TME and Ezh2 inhibition induced PRAME restoration as compared to vehicle controls. IHC for CD3, CD4, FOXP3, and GZMB revealed significant increases in various T-cell populations of the TME, including Tregs and cytotoxic T cells. EZH2 inhibition with EPZ-6438 abrogated these dualistic effects leading to increased immune cell infiltration in the tumor microenvironment and acceleration of apoptosis via PRAME restoration. Moreover, restoration of PRAME antigen presentation by EZH2 inhibition resulted in enhancement of PRAME binding using a T-cell receptor mimic PRAME antibody (Pr20), suggesting immunotherapeutic potential. Conclusion: Our findings highlight multiple functions of PRAME during lymphomagenesis. PRAME restoration by EZH2 inhibition provides a preclinical rationale for synergistic therapies combining epigenetic re-programming with PRAME-targeted therapies. Disclosures Dao: Eureka Therapeutics: Consultancy. Melnick:Jubilant: Consultancy; Epizyme: Consultancy; Constellation: Consultancy; Janssen: Research Funding; Daiichi Sankyo: Research Funding. Scheinberg:Lantheus: Current equity holder in private company; Eureka Therapeutics: Consultancy, Current equity holder in private company, Patents &amp; Royalties: Eureka Therapuetics and MSKCC have filed patent on this ScFV and TCRm; Actinium: Consultancy, Current equity holder in private company; Contrafect: Current equity holder in private company; Sapience: Consultancy, Current equity holder in private company; Iovance: Current equity holder in private company; Enscyse: Current equity holder in private company; Arvenas: Current equity holder in private company; Pfizer: Consultancy, Current equity holder in private company; Sellas: Consultancy, Current equity holder in private company; Oncopep: Consultancy. Scott:Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; NanoString: Patents &amp; Royalties: Named inventor on a patent licensed to NanoString, Research Funding; Celgene: Consultancy; Roche/Genentech: Research Funding. Steidl:Curis Inc: Consultancy; Roche: Consultancy; AbbVie: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy.

Aberrant ERG expression associates with downregulation of miR-4638-5p and selected genomic alterations in a subset of diffuse large B-cell lymphoma.

ERG (avian v-ets erythroblastosis virus E26 oncogene homolog), an oncoprotein in prostate carcinoma and Ewing's sarcoma is associated with poor prognosis in patients with acute myeloid leukemia and T lymphoblastic leukemia. However little is known about ERG in lymphoma. Here we studied ERG in diffuse large B-cell lymphoma (DLBCL) by immunohistochemistry, fluorescence in situ hybridization (FISH), genome-wide microRNA (miRNA) expression profiling, real-time reverse-transcriptase polymerase chain reaction (RT-PCR) and whole exome sequencing (WES). Approximately 30% of de novo DLBCLs (37 of 118) expressed ERG (ERG+). ERG expression showed no significant correlation with DLBCL cell-of-origin classification, patient's age, sex, nodal, or extranodal disease status, tumor expression of p53 or p63. There was no ERG rearrangement in 10 randomly selected ERG+ DLBCLs by FISH. Forty-three miRNAs showed significant differential expression between ERG+ and ERG- DLBCLs. Downregulation of miR-4638-5p was confirmed by real-time RT-PCR. WES not only confirmed known gene mutations in DLBCLs but also revealed multiple novel gene mutations in POLA1, E2F1, PSMD8, AXIN1, GAB2, and GNB2L1, which occur more frequently in ERG+ DLBCLs. In conclusion, our studies demonstrated aberrant ERG expression in a subset of DLBCL, which is associated with downregulation of miR-4638-5p. In comparison with ERG-negative DLBCL, ERG+ DLBCL more likely harbors mutations in genes important in cell cycle control, B-cell receptor-mediated signaling and degradation of β-catenin. Further clinicopathological correlation and functional studies of ERG-related miRNAs and pathways may provide new insight into the pathogenesis of DLBCL and reveal novel targets for better management of patients with DLBCL.

Immunogenomic Landscape of Hematological Malignancies

Understanding factors that shape the immune landscape across hematological malignancies is essential for immunotherapy development. How cancer-cell intrinsic genomic and epigenetic alterations influence immune signatures in hematological malignancies is not known. Here, we integrated over 8,000 transcriptomes of hematologic cancers and multilevel genomic datasets to investigate associations of immune states to cancer molecular subtypes, genetic and epigenetic alterations, and clinical outcomes.We utilized a resource of over 8,000 transcriptomes collected across 36 hematologic malignancies and normal hematopoietic cells (Hemap), together with multi-omics datasets of acute myeloid leukemia (AML) and diffuse large B-cell lymphoma (DLBCL) from The Cancer Genome Atlas and other sources (Figure). In addition to gene expression data, we integrated somatic DNA alterations, methylation data, multiplex immunohistochemistry (mIHC), and flow cytometry to comprehensively map immune-associated features and validate the robustness of the findings. To characterize the composition of the cytolytic immune infiltrate from bulk transcriptomes, we defined a signature of genes most specifically expressed in cytotoxic CD8+ T lymphocytes and natural killer (NK) cells termed cytolytic score.We found significant heterogeneity in the cytotoxic lymphocyte infiltration signature across hematologic malignancies. Highest cytolytic infiltrate was detected in lymphomas and correlated with IFN-γ and myeloid cell infiltration signatures including CXCL9-11 and IDO1, distinguishing the lymphoma microenvironment from leukemias. In addition to transcriptomic microenvironmental properties, specific genetic alterations were associated with cytotoxic lymphocyte infiltration. In DLBCL, driver alterations enriched in the germinal center B-cell like (GCB) molecular subtype including BCL2 translocations and KMT2D were linked to an immune-cold transcriptomic phenotype. In contrast, DTX1 alterations defined immune-infiltrated lymphomas within the GCB molecular subtype. In AML, TP53 mutations and complex karyotype were enriched in a distinct tSNE-based transcriptomic cluster characterized by increased immune infiltration in the bone marrow (BM).Given the importance of effective antigen presentation for adaptive anti-tumor immune responses, we aimed to understand the transcriptional regulation of HLA genes and co-stimulatory and co-inhibitory signaling in subtypes of hematological malignancies. Downregulation of the antigen-presenting HLA II genes was associated with CpG methylation of the promoter region of the HLA class II master regulator CIITA in distinct transcriptomic clusters of AML harboring PML-RARA or NPM1 alterations. Expression of genes encoding immune checkpoint molecules was strongly influenced by the cell-of-origin and microenvironment of each cancer type. We identified novel associations of inhibitory immune checkpoint molecules to disease subtypes, such as VISTA/PD1-H enriched in myeloid malignancies including AML, CML, and MDS, validated by mIHC performed on BM biopsies. Furthermore, variation in the expression of several genes encoding immune checkpoints was associated with somatic mutations (e.g. CD70 in DLBCL), copy-number alterations (e.g. MICB in DLBCL), and DNA methylation (e.g. PDL1 and PDCD1LG2 in AML).Finally, we integrated GTEx gene expression data across tissues to define cancer-germline antigens (CGAs) with an immune privileged tissue expression pattern. CGAs were frequently expressed in multiple myeloma and DLBCL compared to other hematologic malignancies. CGA expression was associated with cytogenetic alterations and increased MYC activity signature in myeloma and CD58 and KLHL6 mutations in DLBCL. In addition, CGA expression in myeloma and DLBCL was linked to reduced antigen gene promoter methylation and decreased survival.In summary, our findings demonstrate that molecular subtypes of hematological malignancies harbor distinct immunological signatures influenced by genetic and epigenetic alterations. Integrating genetic, epigenetic, and transcriptomic data may facilitate the development of precision immune intervention strategies in hematological malignancies. [Display omitted] DisclosuresLeppa:Bayer: Research Funding; Celgene: Consultancy; Roche: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria; Pfizer: Honoraria, Research Funding; Ariad: Research Funding; Novartis: Honoraria, Research Funding.

Recurrent IL4R Somatic Mutations in Diffuse Large B-Cell Lymphoma Lead to an Altered Gene Expression Profile and Changes in Tumor Microenvironment Composition

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is an important and conserved pathway in lymphocytes which is activated by extracellular stimuli such as cytokines and growth factors. Aberrant activation of the JAK-STAT pathway is a hallmark of a variety of lymphomas which leads to increased proliferation/survival as well as immune evasion. Regarding the latter, it has been previously described that activation of the JAK-STAT signaling pathway can alter the secretome of lymphoma cells and the composition of the tumor microenvironment (TME). Specifically, our group reported PTPN1 loss-of-function as well as IL4R gain-of-function (p.I242N) mutations up-regulate the expression of the immune regulatory chemokine CCL17 through a STATs-dependent mechanism in primary mediastinal B cell lymphoma (PMBCL). Here, we assembled a cohort of 340 diffuse large B cell lymphoma (DLBCL) patients uniformly treated with R-CHOP to investigate JAK-STAT signaling mutations (targeted gene sequencing), copy number alteration (SNP arrays), gene expression (RNAseq) and TME composition (Cibersort, IHC). We confirmed the presence of mutations in SOCS1, STAT6 and 9p24 amplification with a frequency of 13.8%, 2.5%, 11.4%, respectively. Interestingly, we also identified the presence of somatic IL4R mutations in DLBCL, including the hotspot p.I242N mutation previously reported in PMBCL. Similarly to what was reported for other alterations in the JAK-STAT pathway, IL4R mutations were significantly enriched in GCB-DLBCL as compared to the ABC subtype, unclassified or double hit lymphomas with DLBCL morphology (p=0.045). Within the GCB group, patients carrying mutations in IL4R showed inferior disease-specific survival (p=0.029) and time to progression (p=0.023) after R-CHOP therapy. Mutational analysis revealed IL4R mutation being significantly concurrent together with mutations in ACTB, KLML6, MYC, STAT6, NLRC5, TNFAIP3 and mutually exclusive with EZH2 mutations (p<0.05). However, among those gene mutations, only mutations in IL4R risk-stratified patients with GCB DLBCL and showed inferior patient outcomes. Moreover, mutations in the extracellular and transmembrane domains of IL4R resulted in gain-of-function leading to constitutive activation of the JAK-STAT pathway in vitro. Gene expression analysis of primary patient samples carrying IL4R mutations displayed increased CCL17 expression (p=0.027), which positively correlated with the level of the T-regulatory marker FOXP3 by IHC (p=0.005). In addition, in silico TME composition analysis revealed a role of IL4R mutations in inducing changes in macrophage polarization. Specifically, we observed an enrichment of an M2-like macrophage phenotype in primary patient specimens carrying IL4R mutations (p=0.01) and a high M2/M1 ratio was significantly associated with inferior patient outcomes (time to progression, log rank p=0.027).In summary, our data suggest a common mechanism between PMBCL and DLBCL where aberrant JAK-STAT activation mediated by mutations in IL4R plays a significant role in altering chemokine expression profiles and TME changes. DisclosuresGascoyne:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Celgene: Consultancy, Honoraria; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Janssen: Research Funding. Steidl:Roche: Consultancy; Seattle Genetics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Tioma: Research Funding; Juno Therapeutics: Consultancy.

Noninvasive Genotyping and Monitoring of Classical Hodgkin Lymphoma

Noninvasive Genotyping and Monitoring of Classical Hodgkin Lymphoma

Using Informatics Tools to Characterize Precision Medicine Treatments for Diffuse Large B-Cell Lymphoma (DLBCL)

▪Diffuse large B-cell lymphoma (DLBCL) is genetically and clinically heterogeneous. Despite immunohistochemical and genomic subtyping, standard firstline DLBCL treatment remains rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). As genetic profiling becomes more accessible and pervasive, studies identifying specific drug-gene interactions in DLBCL are needed to develop future patient-specific DLBCL treatment strategies that improve the delivery of effective and cost-effective, patient-centered care. Methods: We performed a systematic literature review using PubMed Medical Subject Heading and text word terms for DLBCL/genetics and exome sequencing and recorded DLBCL mutations, associated mutation frequencies, pathways, and implications for overall survival (OS) with a focus on recent publications from Reddy et al 2017, Chapuy et al 2018, and Schmitz et al 2018 and related articles. Data for mutation frequencies and OS were extracted from Reddy et al (2017; https://dlbcl.davelab.org/). Mutations were categorized as either favorable or unfavorable. Favorable mutations were associated with 3-year OS ≥ OS for patients equivalent or better than IPI score ≤1 in this dataset (≥90.1%), whereas unfavorable mutations were associated with 3-year OS ≤ OS for patients with an IPI score ≥2 from this dataset (≤64.0%). Potential mutation-specific treatments were identified from the initial literature review, using the Drug-Gene Interaction database (DGIdb, http://www.dgidb.org/, Cotto et al, 2017) and the Open Targets Platform (OTP, https://www.targetvalidation.org/, Koscielny et al, 2017). DLBCL therapeutic groups were defined for mutations/mutation groups that affected ≥5% of DLBCL patients and were associated with targeted therapies based on DGIdb and OTP. Specific drug-gene interactions were characterized using a series of PubMed searches. Results: The analysis identified 32 articles with 55 total therapeutic clusters. 3 mutations were associated with favorable outcomes with an expected 3-year OS of 90.1% (95% CI: 81.1%-95.0%], and 7 mutations were associated with unfavorable outcomes with an expected 3-year OS of 64.0% (95% CI: 56.3%-70.7%) (Figure). The intersection of mutations associated with unfavorable outcomes and mutations associated with targeted therapies yielded 16 therapeutic clusters and revealed a predicted population of 17.6% of DLBCL with poor risk disease and potentially targetable mutations (Table). Drug-gene interaction characterizations identified 267 pertinent studies that included: in vitro studies, mouse models, or clinical trials of the interactions' roles in any cancer. Of these, 7 studies (1 clinical trial, 6 in vitro studies, and 1 mouse model) directly investigated DLBCL specific effects for fostamatinib, ibrutinib, and crizotinib. Conclusions: Linking systematic review with informatics tools identified DLBCL populations, DLBCL specific gene-drug interactions, and potential therapies for trials for patients who relapse and frontline combinations with R-CHOP that can be used in precision medicine strategies for improving outcomes in this disease. DisclosuresFlowers:Genentech/Roche (unpaid): Consultancy; Pharmacyclics/ Janssen: Consultancy; Gilead: Research Funding; Millennium/Takeda: Research Funding; TG Therapeutics: Research Funding; Janssen Pharmaceutical: Research Funding; Karyopharm: Consultancy; Denovo Biopharma: Consultancy; Abbvie: Consultancy; Genentech/Roche: Research Funding; Gilead: Consultancy; Bayer: Consultancy; Celgene: Research Funding; Abbvie: Research Funding; Acerta: Consultancy; Beigene: Consultancy; Pharmacyclics: Research Funding.

Abstract B27: B-cell lymphoma response to anti-CD20 antibodies based therapies is tightly modulated by FOXO1-mediated MS4A1 gene transcription

Abstract No remission or early relapse remains a major clinical concern in diffuse large B cell lymphoma (DLBCL) with a third of patients failing to respond to current regimens or relapsing with resistant disease. Genome and transcriptome sequencing studies have identified FOXO1 as one of the recurrent target for somatic mutations in DLBCL associated with patient short survival. These mutations enhance FOXO1 nuclear localization and activity on target genes. Herein, we determine the role FOXO1 (wild type and mutated) on the expression of the rituximabtarget, CD20. The effect of active mutated FOXO1 on CD20 expression was determined upon inhibition of AKT, exogenous expression of active myristolated AKT, and mutated FOXO1 at its N-terminal region. FOXO1 binding activity to CD20 promoter was assessed using various assays combined with an in silico analysis of a publicly available ChipSeq data. Our results show that the activation of FOXO1 using AKT inhibitors (MK2206 and GDC0068) led to a significant decrease of CD20 transcript and protein levels, which resulted in a significant resistance to rituximab mediated complement dependent cytotoxicity (CDC). Consistently, the overexpression of either wild-type or mutated FOXO1 repressed CD20 expression. Unlike the activity of exogenous wild type FOXO1 that can be inhibited by AKT1, the activity of mutated FOXO1AAA (Thr24, Ser256 and Ser319 mutated to Ala and that cannot be phosphorylated) markedly inhibits CD20 expression. Furthermore, CD20 levels remained high and unchanged by the DNA binding defective mutant of FOXO1H215R, suggesting a FOXO1 mediated regulation of CD20 transcription through FOXO1 binding to CD20 promoter, as confirmed by ChIP, EMSA and the analysis of ChIPSeq data using antiFOXO1 antibody. These results show that FOXO1 mutants that are insensitive to AKT1 kinase activity, effectively suppress CD20 expression in comparison to wild type FOXO1, through a binding to CD20 promoter. In summary, these results establish FOXO1 as an important determinant of cell response to complement-dependent rituximab induced cytotoxicity. In addition, our findings indicate that the genetic status of FOXO1 together with its transcriptional activity need careful attention while designing rituximab-based regimens for the therapy of specific B-lymphomas. Supported by: National Science Center, Poland (NCN#2013/11/B/NZ5/03240), STREAM project (Horizon 2020,#692180). Citation Format: Abdessamad Zerrouqi, Beata Pyrzynska, Michal Dwojak, Nina Miazek, Piotr Zapala, Jakub Golab, Magdalena Winiarska. B-cell lymphoma response to anti-CD20 antibodies based therapies is tightly modulated by FOXO1-mediated MS4A1 gene transcription [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B27.