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T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy characterized by cell subsets and enriched with leukemia-initiating cells (LICs). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of β-catenin in T-ALL, we performed coimmunoprecipitation followed by liquid chromatography–mass spectrometry. Here, we report that a noncanonical functional interaction of β-catenin with the Forkhead box O3 (FOXO3) transcription factor positively regulates LIC-related genes, including the cyclin-dependent kinase 4, which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. In addition, gene expression data at the single-cell level of leukemic cells of primary patients at the time of diagnosis and minimal residual disease (MRD) up to 30 days after the standard treatments reveal that the expression of β-catenin– and FOXO3-dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor ALL. These findings highlight key functional roles for β-catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.

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.

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.

Multivalent antibodies such as sIgA can crosslink motile entities such as sperm and bacteria, creating agglomerates that are too large to permeate the dense mucin matrix in mucus, a process commonly referred to as immune exclusion. Unfortunately, sIgA remains challenging to produce in large quantities, and easily aggregates, which prevented their use in clinical applications. To develop sIgA-like tetravalent antibodies that are stable and can be easily produced in large quantities, we designed two IgGs possessing 4 identical Fab domains, with the Fabs arranged either in serial or in the diametrically opposite orientation. As a proof-of-concept, we engineered these tetravalent IgG constructs to bind a ubiquitous sperm antigen using a Fab previously isolated from an immune infertile woman. Both constructs possess at least 4-fold greater agglutination potency and induced much more rapid sperm agglutination than the parent IgG, while exhibiting comparable production yields and identical thermostability as the parent IgG. These tetravalent IgGs offer promise for non-hormonal contraception and underscores the multimerization of IgG as a promising strategy to enhance antibody effector functions based on immune exclusion.

Background: Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy of mature B cells. The disease has traditionally been subdivided into cell-of-origin (COO) subtypes - germinal centre B cell-like (GCB) or activated B cell-like (ABC) - as determined by expression profiling or immunohistochemistry of the tumor cells. However the role of the immune microenvironment, and how the tumor and immune system interact to influence patient outcomes, remains to be fully investigated. Methods: In this project, we used mass cytometry (CyTOF) to deeply profile both tumor cell phenotypes and the immune microenvironments, alongside ABC/GCB classification and mutation profiling, in a discovery cohort of 54 DLBCL cases. As well, a validation cohort of 129 DLBCL patients were immunologically profiled by high-dimensional conventional flow cytometry, and their immune profiles alongside ABC/GCB classification, mutation profiling, and RNAseq data, were correlated with patient outcomes as measured by progression-free survival (PFS). Results: Analysis of the CyTOF/discovery cohort demonstrated that DLBCL tumor cells are phenotypically unique to each patient, with a small number of samples displaying distinct sub-clonal structure, often distinguished by differential expression of immune-related proteins like MHC-II. ABC/GCB classifications could be recapitulated based on tumor cell phenotypes, demonstrating that while COO was a robust feature, a great deal of heterogeneity exists within these established subtypes. Immunological profiling of the CyTOF/discovery cohort revealed that DLBCL samples could be divided into three distinct groups which roughly correlated with abundances of naïve, activated, or terminally differentiated T cells, respectively. This profiling schema was extended to the validation cohort of 129 patients which in turn led to identification of a subset of patients with a very high risk of disease progression (5-year PFS; 30% high risk vs. 80% low risk, p This final classifier was based on a combination of ABC-DLBCL designation, combined with the presence of an immune microenvironment dominated by terminally differentiated (CD57+) T cells. We performed a limited series of functional studies using primary DLBCL biopsy samples to characterize further these CD57+ T cells as clonally restricted and incapable of responding to antigenic challenge. Interestingly, traditional immune markers of T cell exhaustion such as PD-1, TIM3, LAG3 and TIGIT were not correlated with patient outcomes. Conclusions: Overall, this study demonstrates the utility of immune profiling in risk stratification based on initial diagnostic biopsy material and highlights a subset of DLBCL patients who may benefit from immune-based therapies to rejuvenate the anti-tumor T cell response. We conclude that T cell senescence, rather than exhaustion, is the more relevant feature in DLBCL disease biology and highlights an alternate target for immunomodulatory therapy. Figure 1 Figure 1. Disclosures Craig: Bayer: Consultancy. Slack: Seagen: Consultancy, Honoraria. Scott: Abbvie: Consultancy; AstraZeneca: Consultancy; 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. Steidl: Epizyme: Research Funding; Bayer: Consultancy; Curis Inc.: Consultancy; Seattle Genetics: Consultancy; AbbVie: Consultancy; Trillium Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding.

Follicular lymphoma (FL) is an indolent lymphoma of mature B-cells but may transform to a more aggressive histology, most commonly diffuse large B cell lymphoma. Recurrent mutations associated with transformation have been identified; however, biological predictors to guide initial therapy have remained elusive. We hypothesized that clonal heterogeneity and patient-specific immune responses would contribute to variable clinical outcomes and that understanding the complexity of the entire tumor "ecosystem" would allow more individualized matching of patients with specific therapies. In prior ASH meetings, we presented preliminary analyses of B and T cell-focused phenotypic profiling of 155 newly diagnosed pre-treatment FL biopsy samples at single cell resolution by mass cytometry (CyTOF). These prior analyses unexpectedly revealed two distinct evolutionary trajectories which were independently reflected in both B and T cell compartments. One trajectory expectedly involved germinal center B cells (GCB); however, the other was more related to naïve/memory B-cells (NMB). Interestingly, cluster co-occurrence analysis suggested that the GCB and NMB trajectories were mutually exclusive of another and tended not to be found within the same tumor despite their high prevalences (χ 2 = 29.8, DF=1, p=4.8e-8; χ 2 test). Clustering analysis based on relative abundances of T cell subsets revealed 4 distinct immune patterns: Group 1 was characterized by naive T cells; Group 2 by T follicular helper (Tfh) cells; Group 3 by CD4+ regulatory T (Treg) and CD8 effector memory cells (CD8EM); and Group 4 by a diverse complement of naive, memory, and differentiated effector subsets. We report here further analyses, now incorporating DNA mutational and clinical outcome information. Tumors were parsed into 3 types based on the phenotype of the majority (>50%) of tumor cells present in the diagnostic biopsy: Type A tumors dominated by GCB cells (28% of samples), type B tumors dominated by NMB cells (18% of samples), and type nonA/nonB tumors dominated by neither GCB nor NMB cells (54% of samples). Type A tumors were significantly enriched for mutations in EZH2, TNFRSF14, and MEF2B, while no significant mutational associations were seen in type B and nonA/nonB tumors. Type B was significantly associated with increased risk of transformation, and when combined with a measure of intratumoral phenotypic diversity ("Entropy"), we found that type B tumors with high (above median) Entropy, representing 8.5% of all cases, exhibited a hazard ratio (HR) of 5.9 for transformation risk in comparison to all others combined (log-rank p We also investigated survival outcomes using a sub-cohort of 108 patients who had received bendamustine + rituximab (BR) as their primary therapy. Despite the association of type B tumors with transformation risk, patients with type nonA/nonB tumors exhibited the poorest outcomes as measured by disease-specific survival (DSS; 5yr survival 78% vs 98% for all others combined, log-rank p=0.0241). Combining type nonA/nonB with high Entropy defined 20% of patients with significantly shorter DSS (HR 5.3, log-rank p=0.0019). In multivariate analysis, type nonA/nonB and high risk FLIPI score were significant (p=0.038 and 0.035, respectively), while high Entropy trended with inferior DSS (HR 2.8, 95% CI 0.76-10; p=0.123). Taken together, these data support that CyTOF-defined phenotypic subtypes of FL and intratumoral phenotypic diversity identify clinically significant subgroups at initial diagnosis and compare favorably against FLIPI score in predicting both risk of transformation and inferior DSS. Figure 1 Figure 1. Disclosures Freeman: Incyte: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Amgen: Honoraria; Janssen: Honoraria, Speakers Bureau; Celgene: Honoraria; Teva: Research Funding; Abbvie: Honoraria; Roche: Research Funding. Sehn: Genmab: Consultancy; Novartis: Consultancy; Debiopharm: Consultancy. Savage: BMS: Consultancy, Honoraria, Other: Institutional clinical trial funding; Seattle Genetics: Consultancy, Honoraria; Astra-Zeneca: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Merck: Consultancy, Honoraria, Other: Institutional clinical trial funding; Takeda: Other: Institutional clinical trial funding; Roche: Research Funding; Servier: Consultancy, Honoraria; Beigene: Other: Institutional clinical trial funding; Genentech: Research Funding. Craig: Bayer: Consultancy. Scott: Abbvie: Consultancy; AstraZeneca: Consultancy; Incyte: Consultancy; 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. Steidl: Trillium Therapeutics: Research Funding; Bristol-Myers Squibb: Research Funding; AbbVie: Consultancy; Seattle Genetics: Consultancy; Curis Inc.: Consultancy; Bayer: Consultancy; Epizyme: Research Funding.

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.

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: 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; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

Single-cell RNA sequencing (scRNA-seq) has enabled decomposition of complex tissues into functionally distinct cell types. Often, investigators wish to assign cells to cell types, performed through unsupervised clustering followed by manual annotation, or via “mapping” procedures to existing data. However, manual interpretation scales poorly to large datasets, mapping approaches require purified or pre-annotated data, and both are prone to batch effects. To overcome these issues we present CellAssign (www.github.com/irrationone/cellassign), a probabilistic model that leverages prior knowledge of cell type marker genes to annotate scRNA-seq data into pre-defined or de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while controlling for batch and sample effects. We demonstrate the advantages of CellAssign through extensive simulations and analysis of tumor microenvironment composition in high grade serous ovarian cancer and follicular lymphoma.

Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Introduction: The recently developed DLBCL90 NanoString assay robustly distinguishes primary mediastinal large B-cell lymphoma (PMBCL) from diffuse large B-cell lymphoma (DLBCL), as well as cell-of-origin (COO) subtypes of DLBCL (ABC, GCB, unclassified) and cases with a Double-Hit (DHIT) signature (Ennishi D., JCO 2019). When this assay was applied to biopsies from 343 patients with de novo DLBCL uniformly treated with R-CHOP, nineteen of these cases had a molecular PMBCL signature (mPMBCL), despite the fact that they were diagnosed as DLBCL based on their morphology, immunophenotype and clinical features. Here, we aimed to comprehensively characterize the molecular and clinicopathologic features of these mPMBCL cases. Methods: Survival estimates were calculated using Kaplan-Meier analysis, using time to progression (TTP) and disease specific survival (DSS) as endpoints. We applied whole-exome sequencing, copy number analysis (SNP6.0) and RNAseq to identify somatic mutations, copy number aberrations and differentially expressed genes, respectively. FISH was applied to assess the presence of rearrangements affecting MYC, BCL2 and BCL6. We used data previously obtained within our centre from a PMBCL cohort (n=73) to compare mPMBCL with "bona fide" PMBCL (PMBCL) tumors (Mottok et al, Blood 2019). Results: Median age at diagnosis was significantly higher for mPMBCL compared to PMBCL (62 vs 37 years, p Comparison of the mutational landscape of mPMBCL to PMBCL demonstrated perturbations in the central hallmarks of PMBCL pathogenesis: JAK/STAT signaling, NF-ĸB pathway activation and immune evasion. Genomic aberrations affecting JAK-STAT signaling were shared between mPMBCL and PMBCL, with SNVs or indels affecting IL4R, STAT6 and SOCS1 found in 37%, 37%, and 89% of mPMBCL and 36%, 40% and 69% of PMBCL, respectively. Moreover, copy number analysis revealed JAK2 amplifications in 44% of mPMBCL (71% of PMBCL) and differential gene expression analysis showed increased levels of CD274 (PDL1), PDCD1LG2 (PDL2) and genes belonging to the JAK-STAT-signaling network in mPMBCL. In contrast, these genetic aberrations were rarely observed in a recent whole-exome sequencing study on 304 primary DLBCL tumors (Chapuy B., Nat.Med. 2018). Mutations were also observed in NF-ĸB pathways but the patterns of mutations were distinct between mPMBCL (BIRC3 and BTK) and PMBCL (NKBIE) suggesting convergent biology with alternative mechanisms of pathway dysregulation. Similarly, mPMBCL harbored different mutations (CD83) implicated in immune evasion compared with PMBCL (B2M). Finally, we compared the mutational landscape of mPMBCL with recently described genetically-defined subgroups of DLBCL. Interestingly, a large majority of mPMBCL harbored at least one of the mutations characteristic of "Cluster 4" (incl. CD83, HIST1H1E, SGK1), a subset of DLBCLs defined by Chapuy et al that predominantly includes GCB-DLBCLs. Conclusion: We have identified and characterized a subgroup of DLBCL that expresses the PMBCL gene expression signature. Similar to bona fide PMBCL, these tumors are characterized by genomic aberrations that affect JAK-STAT, NF-ĸB signaling and immune response. However, our data suggest that dysregulation of the latter two pathways is established through distinct evolutionary modes that are reflected in differential mutation patterns and anatomical and clinical presentations. Our findings provide potential novel therapeutic avenues for this subset of lymphoma. Disclosures Sarkozy: Takeda: Research Funding. Savage:BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Scott:Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Juno Therapeutics: Consultancy; Tioma: Research Funding; Roche: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy.

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

Introduction: Grey zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classical Hodgkin lymphoma (cHL), is a rare and poorly defined entity. To decipher its mutational landscape and discover new therapeutic targets, we performed exome sequencing of 31 GZL cases. Methods: GZL cases from the LYSA group (N=139) and BC Cancer (N=30) were centrally reviewed and classified as previously published (Sarkozy et al, Am J Surg Pathol 2019). Whole-exome sequencing was performed on 31 cases with available fresh frozen tissue, using laser micro-dissection (LMD, MMI technology) to enrich for tumor cells and obtain matching normal DNA from microenvironment cells. DNA was extracted (Agencourt® DNAdvance kit) and genomic libraries were constructed with the Ovation ultra-low kit (Nugen®). Exome capture was performed using Agilent SureSelectXT V6+UTR followed by paired-end sequencing (NextSeq®). Somatic nucleotide variants (SNVs) and indels were identified using VarScan, Strelka and Mutect. Parameters affecting the sensitivity and specificity of variant calling were optimized using 7 "gold standard" cases for which DNA from peripheral blood cells was additionally available. Possible oncogenic drivers were identified based on rate of recurrence, MutSigCV and literature review. Results: Among the 31 GZL cases, the median age was 41 y (14-83) with a sex ratio of 15M:16F; 21 cases had mediastinal involvement, including 15 within the thymic area; EBER in-situ hybridization (ISH) was positive in 8 cases. Seven (23%) cases were classified as group-0 (cHL morphology with 100% CD20 expression), 22 (71%) with an intermediate morphology as group-1 (N=9, cHL-like morphology) or group-2 (N=13, LBCL-like morphology) and 2 (6%) as group-3 (LBCL with 100% of CD30 expression). The mean coverage was 96X (42-203) for tumor samples. One case was excluded due to failure in the LMD process. Among the 30 cases, 6628 variants across 4826 genes were found, including 2903 coding mutations (325 indels and 2808 SNVs, mean of 104/sample, range: 15-678), 721 affecting the 5' UTR and 2774 the 3' UTR. A total of 152 genes were identified as being potential oncogenic drivers, with a mean of 11 mutated genes per case (range 2-36). The most recurrently mutated genes were SOCS1 (33%), B2M (23%), GNA13 (20%), LRRN3 (17%), and ZNF217, NCOR1, ITPKB, IRF2BP2, CSF2RB, and CSMD3 (13% each). The epigenetic SWI/SNF and transcription regulation pathway (including NCOR1/2, ARID1A, KMT2D, KMT2A) was affected in 73% of the cases, JAK/STAT in 70% and NF-kB in 19%. As assessed by CNVkit and GISTIC, the most recurrent gains/amplifications identified were in 9p24.1 (JAK2, CD274, PDCD2LG2; 69%) and 2p16.1 (REL, BCL11A; 62%), and losses in 11q14.3 (ATM; 48%) and 12q24.33 (NCOR2; 48%). Based on mutational signature analysis, individual base substitutions were linked to mutagenic processes, with the highest contributions associated with aging (29%) and defective DNA mismatch repair (27%); moreover, mutations attributable to AID/APOBEC activity (5%), were found to be significantly enriched in EBV- vs. EBV+ cases (p = 0.013). EBV+ cases had fewer total variants (mean 98 vs 258, p=0.08) and potential oncogenic variants (mean 7 vs 15, p=0.03) compared to EBV- cases. EBV+ cases also lacked mutations in the NF-kB pathway and MHC-class I components (B2M and HLA-B: 0% vs 43% in EBV-, p=0.06) but had mutations in STAT3, DHX58, ACTB and ATP13A4 (6/7 cases) not present in the 23 EBV- cases. LRRN3 and GNA13 mutations were significantly associated with thymic area involvement (40% vs 0%, p=0.01). Furthermore, fluorescence-ISH indicated that 20% (1/5) of EBV+ cases had a rearrangement in the CIITA locus (16p13.13) vs 53% (9/17) in EBV- cases. Patients with an intermediate morphology had more oncogenic variants than those in group 0 and 3 (mean of 15 vs 6 variants/case, p=0.01 affecting 12 vs 5 genes, p=0.004). Finally, NCOR1 (N=4) and NCOR2 (N=2) mutations were exclusively found in cases with intermediate morphology (23% vs 0% for those with group 0 or 3 morphology). Conclusion: These data suggest that GZL is a highly heterogenous disease harboring somatic driver events shared with PMBCL and HL. We also discovered novel gene mutations pointing to the importance of previously unrecognized pathways in the pathogenesis of GZL. The distinct mutational pattern in EBV+ GZL suggests divergent evolutionary trajectories. Disclosures Sarkozy: Takeda: Research Funding. Salles:Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria; BMS: Honoraria; Amgen: Honoraria, Other: Educational events; Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events. Savage:BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria; Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding. Scott:Celgene: Consultancy; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Juno Therapeutics: Consultancy; Tioma: Research Funding; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Seattle Genetics: Consultancy; Bayer: Consultancy.

Follicular lymphoma (FL) is an indolent disease but transforms in 2% to 3% of patients per year into aggressive, large cell lymphoma, a critical event in the course of the disease associated with increased lymphoma-related mortality. Early transformation cannot be accurately predicted at the time of FL diagnosis and the biology of transformed FL (TFL) is poorly understood. Here, we assembled a cohort of 126 diagnostic FL specimens including 40 patients experiencing transformation (

Primary mediastinal large B cell lymphoma (PMBCL) is a distinct subtype of diffuse large B cell lymphoma thought to arise from thymic medullary B cells. Gene mutations underlying the molecular pathogenesis of the disease are incompletely characterized. Here, we describe novel somaticIL4Rmutations in 15 out of 62 primary cases of PMBCL (24.2%) and in all PMBCL-derived cell lines tested. The majority of mutations (11/21; 52%) were hotspot single nucleotide variants in exon 8 leading to an I242N amino acid change in the transmembrane domain. Functional analyses establish this mutation as gain-of-function leading to constitutive activation of the JAK-STAT pathway and upregulation of downstream cytokine expression profiles and B cell specific antigens. Moreover, expression of I242N mutant IL4R in a mouse xenotransplantation model conferred growth advantagein vivoThe pattern of concurrent mutations within the JAK-STAT signaling pathway suggests additive/synergistic effects of these gene mutations contributing to lymphomagenesis. Our data establishIL4Rmutations as novel driver alterations and provide a strong preclinical rationale for therapeutic targeting of JAK-STAT signaling in PMBCL.

Follicular lymphoma (FL) is an indolent, but incurable malignancy as most patients eventually experience progressive disease. We hypothesized that clonal heterogeneity and patient-specific immune responses would contribute to variable clinical outcomes and that understanding the complexity of the entire tumor "ecosystem" would allow us to better match patients with specific types of tumor- and immune-targeted therapies. In this study, we performed 38-dimensional single-cell phenotyping by mass cytometry (CyTOF) to simultaneously characterize both the substructure of malignant B cell populations as well as the T cell microenvironment in a cohort of 77 diagnostic patient FL biopsies and 35 benign reactive LN (rLN) biopsies. We first applied the t-distributed Stochastic Neighbour Embedding (t-SNE) algorithm to explore intra- and inter- tumoral heterogeneity among malignant B cell populations. t-SNE mapping of individual samples showed that more than a third of FL samples contain at least two phenotypically distinct tumor subpopulations, supporting the notion of multi-clonal tumor architectures presumably due to ongoing clonal evolution. Batched analysis combining all 77 FL cases together with 35 rLN samples revealed two distinct tumor subtypes comprising about 25% (type "A") and 10% (type "B") of total FL samples, respectively, with individual tumors within each subtype showing highly similar and partially overlapping phenotypes. Mapping the same data using Uniform Manifold Approximation and Projection (UMAP), a dimensional reduction algorithm similar to t-SNE but preserves global structure more accurately, revealed that type A tumors localized in close proximity to normal germinal center (GC) B cells, thus fulfilling conventional expectations as to the histogenesis of FL. In contrast, type B tumors localized more closely to pre-GC B cells, implying the existence of an alternate histogenic path in FL. Importantly, we also performed single-cell RNA-Seq on a subset of FL cases which independently confirmed the type A vs type B distinction in whole transcriptomic space. We next analyzed matching T cell data using a modified Statistical Scaffold algorithm in order to place distinct subsets in context with conventionally defined normal T cell populations. Clustering analysis using multi-layer phenograph performed on T cells from all FL and rLN samples combined yielded hundreds of small, but phenotypically distinct populations that were then annotated according to the nearest conventionally defined T cell subset. These imputed designations were used as features to perform hierarchical clustering of samples which revealed 3 major clusters. Cluster1 was characterized by mostly naive T cell populations and contained the majority of rLN samples. Cluster2 was characterized by more differentiated effector T cell populations and was dominated by FL samples. Samples within Cluster2 could be further divided into Tfh, Treg and Th1-rich subgroups. Cluster3 was characterized by a diverse T cell environment including naive, memory and differentiated effector subsets and contained a mixture of rLN and FL samples. Integrative analysis correlating B- and T- cell features revealed type B FL tumors were associated with a Tfh-rich immune landscape. Taken together, these data reveal pervasive phenotypic heterogeneity in both malignant and immune cell compartments of patient FL samples and suggest that defining tumoral subtypes as well as the status of the local immune response within individual samples will support more refined diagnostic classification and highlight functional interactions most amenable to therapeutic targeting. Disclosures Gascoyne: 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:Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Nanostring: Patents & Royalties: patent holding; Bristol-Myers Squibb: Research Funding; Tioma: Research Funding; Roche: Consultancy.

Mechanistic studies in human cancer have relied heavily on established cell lines and genetically engineered mouse models, but these are limited by in vitro adaptation and species context issues, respectively. More recent efforts have utilized patient-derived xenografts (PDX); however, as an experimental model these are hampered by their variable genetic background, logistic challenges in establishing and distributing diverse collections, and the fact they cannot be independently reproduced. We report here a completely synthetic, efficient, and highly reproducible means for generating T-cell acute lymphoblastic leukemia (T-ALL) de novo by lentiviral transduction of normal CD34+ human cord blood (CB) derived hematopoietic progenitors with a combination of known T-ALL oncogenes. Transduced CB cells exhibit differentiation arrest and multi-log expansion when cultured in vitro on OP9-DL1 feeders, and generate serially transplantable, aggressive leukemia when injected into immunodeficient NSG mice with latencies as short as 80 days (median 161 days, range 79-321 days). RNA-seq analysis of synthetic CB leukemias confirmed their reproducibility and similarity to PDX tumors, while whole exome sequencing revealed ongoing clonal evolution in vivo with acquisition of secondary mutations that are seen recurrently in natural human disease. The in vitro component of this synthetic system affords direct access to "pre-leukemia" cells undergoing the very first molecular changes as they are redirected from normal to malignant developmental trajectories. Accordingly, we performed RNA-seq and modified histone ChIP-seq on nascently transduced CB cells harvested from the first 2-3 weeks in culture. We identified coordinate upregulation of multiple anterior HOXB genes (HOXB2-B5) with contiguous H3K27 demethylation/acetylation as a striking feature in these early pre-leukemia cells. Interestingly, we also found coordinate upregulation of these same HOXB genes in a cohort of 264 patient T-ALLs (COG TARGET study) and that they defined a subset of patients with significantly poorer event-free survival (Log-rank p-value = 0.0132). Patients in the "HOXB high" subgroup are distinct from those with ETP-ALL, but are enriched within TAL1, NKX2-1, and "unknown" transcription factor genetic subgroups. We further show by shRNA-mediated knockdown that HOXB gene expression confers growth advantage in nascently transduced CB cells, established synthetic CB leukemias, and a subset of established human T-ALL cell lines. Of note, while there is prior literature on the role of HOXA genes in AML and T-ALL, and of HOXB genes in normal HSC expansion, this is the first report to our knowledge of a role for HOXB genes in human T-ALL despite over 2 decades of studies relying mostly on mouse leukemia and cell line models. The synthetic approach we have taken here allows investigation of both early and late events in human leukemogenesis and delivers an efficient and reproducible experimental platform that can support functional testing of individual genetic variants necessary for precision medicine efforts and targeted drug screening/validation. Further, since all tumors including PDXs continue to evolve during serial propagation in vivo, synthetic tumors represent perhaps the only means by which we can explore early events in cellular transformation and segregate their biology from confounding effects of multiple and varied secondary events that accumulate in highly "evolved" samples. Disclosures Steidl: Seattle Genetics: Consultancy; Tioma: Research Funding; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.

In most human somatic cells, telomeres shorten as a function of DNA replication. Telomere length is therefore an indirect measure of the replicative history of cells. We measured the telomere lengths at XpYp chromosomes in purified human hematopoietic populations enriched for stem cells (Lin−CD34+CD38−Rho−) and successively more mature cells. The average telomere length showed expected length changes, pointing to the utility of this method for classifying novel differentiation markers. Interestingly, the frequency of abruptly shortened telomeres increased in terminally differentiated adult populations, suggesting that damage to telomeric DNA occurs or is not repaired upon hematopoietic differentiation. When Lin−CD34+CD38−Rho− cord blood cells were transplanted into immunodeficient mice, the telomeres of the most primitive regenerated human hematopoietic cells lost approximately 3 kb, indicative of more than 30 cell divisions. Further losses in differentiating cells were similar to those observed in pretransplantation cell populations. These results indicate extensive self-renewal divisions of human hematopoietic stem cells are the primary cause of telomere erosion upon transplantation rather than added cell divisions in downstream progenitors.

Telomeres are essential for genomic integrity, but little is known about their regulation in the normal human mammary gland. We now demonstrate that a phenotypically defined cell population enriched in luminal progenitors (LPs) is characterized by unusually short telomeres independently of donor age. Furthermore, we find that multiple DNA damage response proteins colocalize with telomeres in >95% of LPs but in, Graphical Abstract, Highlights • Normal human mammary gland luminal progenitors (LPs) have very short telomeres • LP nuclei selectively exhibit telomere-associated DNA damage responses • LPs have selectively elevated hTERT expression and telomerase activity • These LP features may play a role in mammary tissue homeostasis and transformation

DNA rearrangements such as sister chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, but they are typically masked by single-cell sequencing techniques. We developed Strand-seq to independently sequence parental DNA template strands from single cells, making it possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On average, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm9 mouse reference genome assembly contains at least 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments have persisted through several iterations of the mouse reference genome and have been difficult to detect using conventional sequencing techniques. The ability to map SCE events at high resolution and fine-tune reference genomes by Strand-seq dramatically expands the scope of single-cell sequencing.

Background: Recent work in both hematologic malignancies and solid tumors has supported the notion that human cancers exhibit marked intra-tumoral heterogeneity (ITH). Results from next generation sequencing (NGS) studies support that subclonal DNA mutations underlie genotypic ITH within a single tumor since the majority of sequence variants are present in only 5-50% of reads for a given tumor sample, and single cell analyses have shown that individual tumor subclones may be ancestrally related in a complex branching hierarchy, suggesting that therapy failures and progressive disease likely arise by Darwinian selection for more aggressive or therapy resistant clones. It has become increasingly clear that it will be important to understand the multi-clonal structure of tumors in order to treat them more effectively. In this study, we sought to use time-of-flight mass cytometry (CyTOF) to explore clonal phenotypic substructure in diffuse large B-cell lymphoma (DLBCL), a diagnostic entity notorious for clinical heterogeneity. Methods: We examined viably frozen single cell suspensions from diagnostic lymph node biopsy samples received for flow cytometric analysis at the BC Cancer Agency. We have thus far acquired CyTOF data from 25 cases of DLBCL using a two-tube, 40-parameter panel encompassing a total of 58 different markers including both surface and intracellular antigens that were selected to reveal heterogeneity within the malignant B-cell population. For each sample acquisition, we included "spiked-in" control cells from pooled reactive (non-malignant) lymph node samples to control for staining variation between antibody/reagent lots and also run-to-run CyTOF instrument drift, facilitated by a CD45 antibody "barcoding" approach. We analyzed the data using a combination of viSNE, Isomap, and PhenoGraph analysis packages. Results: Analysis of individual tumor samples readily distinguished between malignant and residual normal B-cell populations, and also revealed distinct subpopulations among malignant cells of varying degrees of relatedness to one another. These subpopulations were then sorted from one another by conventional FACS from parallel vials of cryopreserved cells using lower dimensional sorting strategies derived from the 40-parameter CyTOF data. Sorted subpopulations will be analyzed by targeted amplicon sequencing for single nucleotide variants identified from whole exome sequencing data obtained from unsorted material to explore the hypothesis that these may represent genotypic subclones. Analysis of multiple tumor samples at once yielded several observations. First, B-cells from reactive lymph nodes and non-malignant B-cells within patient lymphoma specimens reproducibly cluster atop one another, indicating highly similar if not identical phenotypic profiles. Second, the majority of patient DLBCL tumors form cohesive individual clusters, separate and distinct from one another, suggesting that the 40-dimensional panel defines cell populations with sufficient resolution such that each patient's tumor can be uniquely identified. Third, individual DLBCL tumors do not aggregate in tight proximity with one another to the extent that we observe among patient follicular lymphoma (FL) samples, suggesting DLBCL represents a broader diversity of phenotypic classes. Fourth, there is local, but loose aggregation of ABC versus GCB subtypes, but there are also clear outliers and areas of intermingling between ABC and GCB tumors, as defined by immunohistochemistry. Finally, a subset of DLBCL tumors exhibit minor subpopulations that map apart from their corresponding "parent" tumor populations, but yet overlap one another, raising the possibility of divergent evolution away from (or alternatively convergent evolution towards) a common tumor archetype. Conclusions: Taken together, these observations support that novel information can be derived from CyTOF data with important implications for our understanding of both intra- and inter-tumoral heterogeneity in DLBCL. Disclosures Scott: Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Introduction: Constitutive MHC class II expression is a hallmark of antigen-presenting cells, including B cells, and is indispensable for the initiation of antigen specific immune responses. It has been shown that certain B cell lymphoma entities are able to evade immune recognition by downregulation of MHC molecules on the tumor cell surface. We have previously identified recurrent chromosomal rearrangements of CIITA, the master regulator of MHC class II transcription, as one possible mechanism to reduce MHC class II expression in primary mediastinal large B-cell lymphoma (PMBCL) and classical Hodgkin lymphoma (cHL) (Steidl et al., Nature 2011). Furthermore, we have recently described a 1.6kb breakpoint cluster region within intron 1 of CIITA and have shown in a small sample set of PMBCL cases that deletions, insertions and single nucleotide variants (SNV) are commonly found within this genomic region (Steidl, ASH abstract # 437, 2011). Therefore, we aimed to explore the frequency of these alterations and the correlation with CIITA and MHC class II protein expression in a larger cohort of PMBCL cases and to further characterize their functional significance. Methods: We have comprehensively analyzed 45 diagnostic PMBCL samples for the presence of coding sequence mutations as well as alterations within the promoter III region and the first 3kb of intron 1 using deep amplicon sequencing (Illumina TruSeq) and/or Sanger sequencing. In addition, we characterized the PMBCL-derived cell lines U2940 and Med-B1 by whole transcriptome paired-end sequencing (RNA-seq). To elucidate the functional consequences of the coding sequence mutations identified in these two cell lines we performed retroviral transductions of wild type CIITA and CIITA mutants in a CIITA and HLA-DR expression-negative cell line (DEV, nodular lymphocyte predominant Hodgkin lymphoma-derived). We subsequently analyzed CIITA mRNA expression using qRT-PCR and HLA-DR surface expression using flow cytometry. Furthermore, we applied immunohistochemistry (IHC) to determine expression levels of CIITA and HLA-DR in a large cohort of PMBCL samples represented on two tissue microarrays (TMA, n=149). The TMAs were also used for fluorescence in-situ hybridization (FISH) to evaluate the presence of copy number alterations or translocations of the CIITA locus. Results: FISH was interpretable in 115 samples with a CIITA break-apart (CIITA-ba) frequency of 33.9% (39/115). Correlative analyses revealed that decreased CIITA protein expression by IHC was significantly correlated with the presence of CIITA-ba (P=0.019), whereas HLA-DR expression was not correlated with CIITA-ba status alone (P=0.219). However, we could demonstrate a positive correlation between protein expression of CIITA and HLA-DR (Pearson r=0.45, P Using RNA-seq, we have detected two missense mutations in the Med-B1 cell line affecting both alleles in functionally relevant protein domains. Furthermore, we identified a novel NUBP1-CIITA fusion transcript in U2940 also harboring an SNV on the other allele resulting in the transcription of an elongated protein due to the loss of the original stop codon. Ectopic expression of these CIITA mutants in DEV, which has been shown to have undetectable levels of CIITA and HLA-DR due to a biallelic CIITA inactivation, revealed that these individual SNVs showed a diminished capability to restore HLA-DR surface expression in comparison to wild type CIITA as measured by flow cytometry. Conclusions: Here we show that the presence of CIITA rearrangements is significantly associated with low CIITA protein levels, and we could demonstrate that protein expression of CIITA and HLA-DR are positively correlated in PMBCL. Furthermore, CIITA is frequently targeted by coding sequence mutations and intronic deletions in PMBCL cell lines and clinical samples. Functional studies demonstrate that genomic alterations in CIITA contribute to downregulation of MHC class II expression in malignant lymphomas and therefore represent a potent mechanism of acquired immune privilege and escape from immune surveillance. Disclosures No relevant conflicts of interest to declare.

The loss of telomeric DNA with each cell division contributes to the limited replicative lifespan of human T lymphocytes. Although telomerase is transiently expressed in T lymphocytes upon activation, it is insufficient to confer immortality. We have previously shown that immortalization of human CD8+ T lymphocytes can be achieved by ectopic expression of the human telomerase reverse transcriptase (hTERT) gene, which encodes for the catalytic component of the telomerase complex. To study the role of endogenous hTERT in the lifespan of human T cells, we blocked endogenous hTERT expression by ectopic expression of dominant-negative (DN) hTERT. Cells expressing DN-hTERT had a decreased lifespan and showed cytogenetic abnormalities, including chromosome ends without detectable telomeric DNA as well as chromosome fusions. These results indicate that while endogenous hTERT cannot prevent overall telomere shortening, it has a major influence on the longevity of human T cells. Furthermore, we show that up-regulation of hTERT in T cells upon activation decreases over time in culture. Long-term–cultured T cells also show a decreased expression of c-myc upon activation, resulting in less c-myc–induced transcription of hTERT. Moreover, memory T cells, which have expanded in vivo upon antigen encounter, expressed a lower level of hTERT upon activation than naive cells from the same donor. The observed inverse correlation between telomerase levels and replicative history suggests that telomerase levels in T cells are limiting and increasingly insufficient to sustain their proliferation.

We have previously reported the cloning of the Hematopoietic Pbx Interacting Protein (HPIP), a novel protein discovered through its interaction with Pbx1. HPIP is expressed in early hematopoietic precursors, can bind all members of the Pbx family and can inhibit the transcriptional activation of the oncogene E2A-Pbx. To further understand the function of HPIP, we have analysed its cellular localization and characterized its functional localization domains. Using fluorescence microscopy to follow the distribution of different HPIP sequences fused to GFP, we found that HPIP localizes predominantly to cytoskeletal fibers but has the potential ability to shuttle between the nucleus and the cytosol. The cytoskeletal localization of HPIP is mediated by an N-terminal leucine rich region (between aa 190-218) and can be disrupted by the microtubule destabilizing drug vincristine. The HPIP C-terminal domain (aa 443-731) bears a nuclear export activity that is blocked by the CRM1 inhibitor Leptomycin B. In addition, we found two basic amino acid regions located between aa 485-505 and aa 695-720 that contain nuclear import activities attenuated by nuclear export. These observations support a model in which the constitutive attachment of HPIP to the cytoskeleton could be modified by changes in functional domains implicated in nuclear export, import and cytoskeleton binding sequences, allowing the molecule to shuttle between the nucleus and the cytosol.