Citations

Abstract Background and Aims. Despite the fact that the myeloma mutational landscape was elucidated several years ago, there are yet no approved precision therapies targeting specific driver mutations. The role of common, but somewhat enigmatic, mutational events remains elusive, for example, the loss-of-function mutation of the RNA-processing exosome ribonuclease, DIS3, the third most-common mutation after KRAS and NRAS mutations. Furthermore, complex clonal architectural patterns (such as subclonal BRAF mutations nested within RAS-mutant clones) complicate the design of targeted therapy trials. To generate precision in vivo models that recapitulate the complex clonal and mutational architecture of myeloma, we integrated two powerful technological platforms: the first RAS-driven myeloma in vivo model (VQ model) created through a collaboration between the Zhang and Asimakopoulos labs coupled with flexible avian leukosis virus (ALV)-mediated gene transfer that permits myeloma genome editing in a temporally- and spatially- controlled fashion (Prdm1: TVb-mRFP model)(Asimakopoulos and Varmus, J Virol, 2009; 83(10):4835-43). VQ is based on the state-of-art Vk*MYC model (Chesi et al., Cancer Cell 2008;13(2):167-80). VQ was generated by conditionally activating an NrasQ61R allele in Vk*MYC mice. The resultant VQ model recapitulates an advanced, proliferative and drug-resistant phase of disease characterized by RAS mutations. The transducibility of VQ restored the functionality of the ALV-gene transfer technology. This technology exploits the fact that avian retroviruses cannot transduce mammalian cells unless they are engineered to ectopically express an avian retroviral receptor (in this case, the receptor TVb, driven by a plasma-cell-specific regulatory elements). Recently, the ALV gene transfer technology was adapted to achieve CRISPR-Cas9 mediated gene editing in the tumor niche in vivo (Oldrini et al., Nat Comm 2018;9(1):1466). Approach. We generated TVb-expressing VQ lines (CD138+ B220neg) that home to the bone marrow upon transplantation into Prdm1:TVb-mRFP fully immunocompetent syngeneic recipients in the C57BL6/J background. These VQ cells were engineered to express the nuclease Cas9. To determine the efficiency of gene transfer through ALV vectors (as a prelude to sgRNA delivery), pilot experiments were conducted using two types of ALV vector carrying a fluorescent reporter, GFP (green fluorescent protein): a) an RCAS-type vector that drives reporter expression from the native ALV LTR and b) an RCAN-type vector that drives reporter expression from an internal CMV promoter. Results. ALV titers achieved were routinely in excess of 107-108 infectious units/mL. Parental Vk*MYC cells expressing the avian receptor TVb were poorly transducible with ALV vectors. By contrast, VQ cells expressing TVb were readily transducible. At multiplicity of infection (MOI)=2, 68% of VQ myeloma cells expressed reporter fluorescence after a single round of transduction. At MOI=5 the transduction rate rose to 80%. The mean fluorescence intensity (MFI) positively correlated with MOI, consistent with higher-copy proviral integration at higher MOI. At equivalent MOI, GFP fluorescence from RCAS vectors was more intense than GFP fluorescence driven by an internal CMV promoter in RCAN vectors, suggesting that the ALV LTR is a stronger promoter than CMV in VQ myeloma cells. 100% transduced VQ cells were CD138+B220neg at all conditions tested. Conclusions and Current Focus. We are using ALV-CRISPR technology to generate precision myeloma models to support the next wave of personalized therapy and immunotherapy for this recalcitrant and incurable cancer. Progress on three in vivo models under construction will be presented at the meeting:Subclonal Braf mutation nested within clonal Nras mutation. These models will be treated with next-generation RAF inhibitors (Karoulia Z et al.Nat Rev Cancer 2017;17(11):676-91).Dis3 loss-of function mutation in constellation with co-clonal Ras mutation. The pathogenic consequences of Dis3 disruption remain poorly understood as no parallels exist in other cancers.The murine homolog of the t(4;14) translocation. We are targeting breakpoint cluster regions within perfectly syntenic sequences on mouse chr. 5 (containing Fgfr3 and Mmset loci) and mouse chr.12 (containing the murine Igh locus). Disclosures No relevant conflicts of interest to declare.

Abstract T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule that is expressed on a variety of cell types including NK cells, effector and memory T cells, and Treg cells. Upon ligation with CD155, TIGIT delivers an inhibitory signal and negatively regulates anti-tumor responses. While important in normal T-cell biology, the pathological significance of TIGIT expression and function in the tumor microenvironment of the patients with follicular lymphoma (FL) is largely unknown. The present study sought to phenotypically and functionally characterize TIGIT+ T cell subsets in FL. While its expression is not detected on resting T cells in peripheral blood, we found that TIGIT is highly expressed on intratumoral T cells from FL. Treatment with cytokines such as IL-4 and TGF-β downregulated TIGIT expression on T cells. We found that TIGIT is predominantly expressed on effector memory T cells (TEM) with an activation/exhausted phenotype, and TIGIT+ T cells have higher expression levels of CD69 and PD-1 when compared to TIGIT- T cells. Functionally, TIGIT+ T cells displayed reduced capacity of cell proliferation and cytokine production (IFN-γ and IL-2). Using mass cytometry (CyTOF), we observed that TIGIT is abundantly expressed on some intratumoral Treg (CD4+CD25+Foxp3+) cells, while other Treg cells lack TIGIT expression in FL, forming two subsets of Treg cells: CD4+CD25+TIGIT+ and CD4+CD25+TIGIT-. These two subsets are phenotypically distinct in that CD25+TIGIT+ cells exhibited increased expression of activation/costimulatory markers such as CD69, CD27 and CD28 compared to CD25+TIGIT-, suggesting an activated Treg subset of CD25+TIGIT+ cells. This CD25+TIGIT+ subset had increased suppressive function and could more effectively inhibit activation and proliferation of CD8+ T cells than CD25+TIGIT- cells. Furthermore, we found that lymphoma B cells promoted the development of TIGIT-expressing T cells as TGF-β-mediated downregulation of TIGIT on T cells was inhibited when CD19+ lymphoma cells were present. Using a cohort of 31 FL patients, we found that intratumoral TIGIT-expressing T cells were associated with a favorable prognosis. Patients with TIGIT+ T cell numbers greater than 50% had better overall survival than patients with TIGIT+ T cell numbers less than 50%. Taken together, our results reveal a role of TIGIT in defining Treg cell subsets with different immune function and TIGIT expression may be useful in predicting patient outcome in FL. Disclosures Ansell: Takeda: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer: Research Funding; Seattle Genetics: Research Funding; Celldex: Research Funding; Regeneron: Research Funding; LAM Therapeutics: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Merck & Co: Research Funding.

Abstract Multiple myeloma (MM) is characterized by clonal expansion of malignant plasma cells (PCs) and aberrant production of monoclonal immunoglobulin detected as an M spike using serum protein electrophoresis. In the United States, MM represents ~15% of hematologic malignancies and is one of the few cancers increasing in incidence (e.g., 14,400 in 1996 to 30,770 in 2018, from SEER). Previously, a Vk*MYC mouse model was described, in which AID-dependent activation of MYC transgene in germinal center (GC) B cells catalyzes a highly penetrant, indolent MM after a prolonged latency, suggesting that additional genetic mutations are required for the malignant MM progression. Recent sequencing of paired tumor/normal samples from advanced or refractory MM patients identified that constitutive activation of Ras signaling pathway (KRAS: 23%; NRAS:20%; BRAF: 8%) associates with MM progression and therapy resistance. To determine whether oncogenic Nras promotes the progression of Myc-induced indolent MM to a malignant stage, we generated NrasLSL Q61R/+; Vk*MYC; IgG1-Cre (VQ) mice along with single mutant mice Vk*MYC; IgG1-Cre or NrasLSL Q61R/+; IgG1-Cre. To boost NrasQ61R expression in GC B cells, 6-7 weeks old mice were immunized with NP-CGG. A significant fraction of VQ mice developed M-spike after immunization and subsequently died of a highly aggressive MM, which was characterized by high proliferative index, hyperactivation of AKT and ERK pathways, and disease hallmarks (e.g. osteolytic lesions, anemia, and kidney injury). VQ myeloma was readily transplantable into serial syngeneic recipients. In our preliminary study, combined Bortezomib and AZD6244 treatment attenuated MM phenotypes and prolonged the survival of VQ recipients. To facilitate easy molecular or genetic manipulations of VQ MM cells, we established two cell lines from primary cells. These cells could be easily infected (see another abstract from our co-author, Dr. Asimakopoulos's group). The cultured MM cells also express high levels of CD155, a ligand of immune checkpoint TIGIT, which has been recently reported to mediate a significant immune checkpoint blockade in MM patients. We further investigated the transcriptional signature of VQ MM cells (from bone marrow [BM] and from lymph nodes [LN]) vs wild-type (WT) plasma cells using the Illumina Bio-Rad's Single-Cell Sequencing platform. Illumina's Sure Cell app was used to de-multiplex samples, process barcodes and perform single-cell 3' gene counting. The processed data were then analyzed using Seurat R package to yield cluster information and their associated enriched genes. Our results revealed distinct clusters of CD138+ MM and WT cells, and also showed high similarity & overlap between BM & LN MM samples. Consistent with the genetic changes introduced to VQ MM cells, both Myc and Nras expression levels were significantly elevated in MM clusters compared to WT clusters. We also found that transcriptional levels of several potential therapeutic targets were significantly elevated in MM clusters, including Integrin alpha 4 and metabolic enzymes. Currently we are validating the scRNA-Seq results. Taken together, we generated a novel mouse model in which activation of Vk*MYC and oncogenic Nras in GC B cells results in a highly malignant, transplantable MM. The VQ mouse model represents an important innovation that will serve as a platform to investigate pathogenesis of resistant and refractory multiple myeloma and allow for testing the efficacy of novel therapeutic agents. Disclosures No relevant conflicts of interest to declare.

Abstract Through the recent elucidation of molecular and cellular processes in multiple myeloma (MM) pathogenesis that effect well-defined proliferative and survival pathways, a number of molecular targets for anti-cancer agents have emerged. This includes proteins involved in cell cycle regulation. A recent model of early MM pathogenesis proposes that at least one of the cyclin D genes is dysregulated in all myeloma tumors facilitating activation of cyclin dependent kinase (CDK)4 (or CDK6), and transition from G1 to S phase. It is hypothesized that this renders myeloma cells more responsive to bone marrow (BM) derived proliferative stimuli including IL-6 that upregulates MYC expression in MM cells and cooperates with cyclin D to promote transit to S-phase. In addition, recent studies have established a causative role of MYC dysreguation in progression from monoclonal gammopathy to myeloma. Thus an approach targeting deregulated cell cycle progression and MYC may prove effective MM therapy. Purvalanol is a potent and selective inhibitor of CDK1, an important regulator of cell cycle progression and has been reported to induce MYC-dependent apoptosis. Activity of purvalanol against a panel of 14 standardized, annotated myeloma cell lines was measured in a 48 hour MTT viability assay. IC50-s of all 14 cell lines ranged between 5 uM to 7.5 uM. Western blot and real-time PCR analysis revealed variability of MYC protein and mRNA levels between the 14 myeloma cell lines. We compared potential therapeutic activity of purvalanol against 3 myeloma cell lines (U266, H929, KMS12PE) with low, intermediate, and high levels of c-MYC expression, respectively. All three cell lines demonstrated G2-M growth arrest however marked apoptosis as determined by PI/annexin V staining was observed only in the cell lines with the highest level of MYC expression. Exposure of MM patient-derived BM mononuclear cells to purvalanol preferentially induced apoptosis of CD138+ MM cells. In contrast, purvalanol was minimally cytotoxic to the non-myeloma cell fraction and to non-transformed fibroblast cell lines (MRC5, IMR90 and W138) and failed to inhibit normal bone marrow-derived CD34 colony formation. Recent studies have demonstrated that the BM microenvironment offers protection of myeloma cells from chemotherapeutic agents by common mechanisms. Myeloma cell lines were cultured in 3 different conditions to mimic the tumor’s protective microenvironment. Soluble factors produced by the BM, IL6 and IGF-1 induced a modest degree of resistance to purvalanol while co-culture on BM stroma cells was completely protective. Studies evaluating the in vivo efficacy of purvalanol in a novel Vk*myc transgenic mouse model that spontaneously develops myeloma with a low proliferative index are ongoing and will be presented. The CDK1 inhibitor, purvalanol demonstrates broad single agent activity against myeloma cells with enhanced activity against MYC overexpressing cells. However, the strong protective effect of the BM microenvironment suggest that combination with agents that can reverse cell-adhesion mediated drug resistance may prove beneficial in optimizing the efficacy of this class of drugs for the treatment of MM.

Abstract Background: B-cell non-Hodgkin lymphomas (NHL) are common lymphoid malignancies in which malignant B-cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. The microenvironment that supports the growth and survival of NHL B cells consists of a complex network of immune cells and cytokines and its specific composition has been shown to have significant clinical implications. The intratumoral immune cells include effector and regulatory T (Treg) lymphocytes that are more than simple residual elements from the normal lymph node structure. We previously explored whether the extent of T cell infiltration played a role in the clinical outcome of patients with B-cell NHL and found that an increased percentage of CD4+ T cells in the diagnostic biopsy of lymphoma patients significantly correlated with an improved 5-year overall survival. We have also shown that intratumoral Treg cells significantly suppress the anti-tumor response. Based on the fact that intratumoral T-cells are important in B-cell NHL, we evaluated whether genetic variation in genes that regulate the T-cell response may be associated with lymphoma risk. Methods: We genotyped 257 single nucleotide polymorphisms (SNPs) from 50 candidate genes related to T-cell differentiation and function in a clinic-based study of 441 Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. Tagging and nsSNPs were selected from HapMap. The most prevalent homozygous genotype was used as the reference group and each SNP was modeled individually as having a log-additive effect, expressed as ordinal odds ratios (OR) per variant allele and 95% confidence intervals, in an age- and sex-adjusted logistic regression analysis. We also evaluated the consistency of the findings for the most common types of B-cell NHL - diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and CLL/SLL. For gene-level analyses, we used principal components (PC) analysis using all SNPs from a gene; those PCs that explained > 90% of the variability were then modeled in a logistic regression analysis and significance was determined using a likelihood ratio test. We also evaluated the association of haplotypes from each gene with risk of NHL using the score test implemented from HAPLO.SCORE. Results. The mean age at diagnosis was 60.1 years for cases and 58% were male; among controls, the mean age at enrollment was 61.7 years and 55% were men. In the PC gene analyses, PRF1 (perforin gene - involved in cytotoxicity; p=0.004), CD276 (B7-H3 gene - involved in co-stimulation; p=0.01), TBX21 (T-bet gene – regulates Th1 cell development; p=0.02), and IL6 (p=0.02) were significant at p<0.05; haplotype analysis showed similar results, with the addition of CARD15 (NOD2 gene – regulates Th1 responses; p=0.02). There were 2 SNPs in PRF1, and both the intronic SNP rs3758562 (OR=1.38; 1.13–1.69) and the synonymous coding SNP rs885821 (OR=0.80; 0.63–1.02) were associated with NHL risk overall and for each subtype. There were 2 SNPs in CD276, and both the intronic SNP rs7176654 (OR=1.29; 1.07–1.56) and the mrna-utr SNP rs3816661 (OR=0.82; 0.68–0.99) were associated with NHL risk overall and with the CLL/SLL and follicular NHL subtypes specifically. There were 2 SNPs in TBX21, and only the mrna-utr SNP rs7502875 (OR=0.73; 0.59–0.92) was associated with NHL risk overall and for each subtype. There were 11 SNPs in IL6, and the intronic SNP rs2069835 (OR=1.88; 1.24–2.84) and the locus-region SNP rs2069824 (OR=1.73; 1.20–2.50) were each associated with NHL risk overall and each subtype. Finally, there were 9 SNPs in CARD15, with only the nonsynonymous coding SNP rs5743291 (OR=1.43; 1.02–1.98) being associated with overall NHL risk, and risk was specific to FL. Conclusions. Genetic variation in several genes that play critical roles in T-cell maturation and function was associated with lymphoma risk. These genes appeared to be associated with the differentiation and function of effector T-cells particularly Th1 and Th17 cells. These results provide potentially important insights into the role of T-cells in lymphomagenesis.

Abstract Background: Cutaneous T-cell lymphoma (CTCL) is an incurable lymphoproliferative disorder characterized by the clonal expansion of malignant CD4+ T cells. Currently there is a poor understanding of the underlying mechanisms in the development and progression of this disease. Members of the TNF superfamily have been shown to be important mediators in the formation and regulation of normal T cell responses. A proliferation-inducing ligand (APRIL), a newly identified member of the TNF superfamily, has been shown to stimulate tumor cell growth as well as proliferation of primary B and T lymphocytes. APRIL transcript levels are reported to be low in normal tissues with the highest levels found in peripheral blood leucocytes. In contrast, higher mRNA levels have been detected in tumor cell lines and primary tumor tissues and various tumor cell lines have been shown to respond to APRIL with increased tumor cell proliferation and decreased apoptosis. The striking effects of APRIL on tumor cell growth and survival raise the possibility that the APRIL and its receptors-TACI and BCMA may be involved in the pathogenesis and maintenance of T cell malignancies. Goal: To explore the involvement of APRIL on normal and malignant T cell growth and survival. Results: Under resting conditions, there was minimal APRIL binding to normal T-cells and no TACI or BCMA expression. However, stimulation of resting T cells with OKT3 (anti-CD3) or a combination of PMA and ionomycin resulted in an increase in APRIL binding and TACI expression on a subset of activated T-cells. When CD3+ resting human T cells were cultured in the presence of APRIL, IL-2, or OKT3 alone or a combination, normal T cells had greater viability compared to the nil control and APRIL mediated-T cell survival was similar level to that seen with IL-2 or OKT3 alone. CSFE labeled T cells were activated with suboptimal dose of OKT3 and incubated in the presence of APRIL and /or IL-2. Both APRIL and IL-2 alone induced cell proliferation, while treatment with the combination of APRIL and IL-2 had a synergistic effect on T cell proliferation. Malignant T cells isolated from the blood of patients with Sezary Syndrome exhibited a similar pattern of APRIL binding and APRIL also enhanced their survival. Gene array experiments were performed to identify genes involved in APRIL-mediated survival. In normal and primary malignant T cells the anti-apoptotic genes BCL2A1, BIRC6, CHK1, MCL-1, and MDM2 were upregulated and the pro-apoptotic genes CASP5, DR5, and TNFR2 were downregulated in response to APRIL. Conclusion: APRIL promotes the survival of normal and malignant T cells and co-stimulates CD3-mediated T cell proliferation. The gene array experiments suggest that APRIL regulates the expression of anti-apoptotic and pro-apoptotic genes in both normal and malignant T cells. Taken together, these data indicate an important role for APRIL in the biology of normal T cells and in the pathophysiology of T cell lymphoma.

Abstract Background: Patients with newly diagnosed, advanced-stage, follicular B-cell non-Hodgkin’s lymphomas (NHL) commonly receive rituximab as initial therapy. While antibody dependent cytotoxicity (ADCC) is felt to be the predominant mechanism of action for rituximab, the immunologic effects of rituximab are largely unknown. We have previously shown that genetic polymorphisms in IL-8, IL-2, IL-12 and IL-1 receptor antagonist (IL-1RN) have prognostic significance in follicular lymphoma. The goal of this study was to measure changes in the abovementioned and other serum cytokine levels in patients receiving single agent rituximab as initial therapy for follicular NHL and to identify cytokines that are associated with an increased risk of disease relapse. Methods: Patients with untreated follicular grade 1 NHL, and measurable stage III/IV disease were treated with rituximab 375 mg/m2 intravenous weekly for 4 doses and were then followed for response and time to progression (TTP); no maintenance therapy was provided. Thirty-seven patients were enrolled in this clinical trial between July 1999 and May 2001. As previously reported, the overall response rate in the 36 eligible patients was 72%, with 36% complete remissions. Now, with a median follow up of 5.1 years, the median TTP is 2.2 years (95% CI, 1.2–3.3 years) and the 5-year overall survival is 77% (95% CI: 64–92%). We measured the serum levels of a variety of inflammatory cytokines by ELISA at baseline and 1 month after completing treatment. Serum specimens were available in 30 patients. Thirty two cytokines or receptors including IL-1β, IL-1RN, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p40/p70, IL-13, IL-15, IL-17, TNF-α, IFN-α, IFN-γ, GM-CSF, MIP-1α, MIP-1β, IP-10, MIG, Eotaxin, RANTES, MCP-1, VEGF, GCSF, EGF, FGF-basic, HGF, BLyS and APRIL were analyzed. Results: A significant increase was seen in the serum levels of BLyS (p=0.0001) after rituximab therapy, while IL-12 (p=0.0002), IL-1RN (p=0.0001), IL-2R (p=0.0001), IL-1β (p=0.02), IL-7 (p=0.02), IL-10 (p=0.02), and MIG (p=0.05) all significantly decreased. All other cytokines/receptors remained unchanged. Higher pretreatment serum levels of IL-2R (p=0.008) and IL-12 (p=0.001) correlated with a shorter TTP. Furthermore, elevated serum IL-2R levels pretreatment correlated with a lack of response to rituximab (p=0.03). Differences in overall survival could not be determined due to the fact that only 8 patients enrolled in the study have died. Because serum IL-2R levels correlated with response and TTP, we measured the cell surface expression of IL-2R in the tumor by flow cytometry. IL-2R was detected on a subpopulation of CD4+ T-cells in all specimens tested (n=14), and on malignant B-cells in 5 cases (36%). Conclusions: Pretreatment serum levels of IL-2R and IL-12 are important prognostic factors for response to therapy or TTP in follicular lymphoma patients treated with rituximab. Furthermore, serum levels of BLyS, IL-1RN, IL-7, IL-10, IL-1β and MIG also change after rituximab treatment. Targeting these cytokines may therefore provide additional clinical benefit to patients treated with rituximab.

Abstract Numerous clinical therapies have attempted to modulate tumor cell immunity, but for the most part, have proven unsuccessful. The inability to produce or augment an effective immune response is due in part to regulatory T (Treg) cells, which inhibit CD4 and CD8 T cell function. Our group has recently shown that Treg cell numbers are elevated in NHL tumors and that NHL B cells induce the development of Treg cells thereby inhibiting anti-tumor responses. The ability of NHL B cells to direct the cellular composition of their microenvironment is critical to our understanding of tumor immunity and we therefore wanted to determine if NHL B cells also directed the expansion or reduction of other T cell populations. IL-17-secreting CD4+ T cells (TH17), a newly characterized CD4+ T helper cell lineage, promote inflammation and play an important role in autoimmune disease. IL-17 has been shown to inhibit tumor cell growth suggesting a potential role for TH17 cells in anti-tumor immunity. We therefore set out to determine if TH17 cells were present in NHL tumors and whether or not their numbers were regulated by NHL B cells. Using unsorted mononuclear cells from malignant lymph nodes, we were unable to detect IL-17 expression in resting CD4+ T cells or CD4+ T cells activated with PMA/Ionomycin stimulation (less than 1%). However, IL-17-secreting CD4+ T cells could be detected in significant numbers in inflammatory tonsil and normal PBMCs. Interestingly, depletion of CD19+ NHL B cells from mononuclear cells obtained from patient biopsies resulted in detection of a clear population of IL-17-secreting CD4+ T cells (5%). These results suggest that NHL B cells suppress TH17 cell differentiation. The frequency of IL-17-secreting CD4+ T cells could not be further enhanced by the addition of exogenous TGF-b and IL-6, a cytokine combination favoring for TH17 differentiation, suggesting a further impairment of TH17 cell differentiation in the tumor microenvironment. In contrast, Foxp3 expression could be detected in resting CD4+ T cells (30%) and could be induced in CD4+CD25−Foxp3− T cells activated with TCR stimulation (28%). Contrary to the inhibition of TGF-b-mediated TH17 differentiation, Foxp3 expression could be dramatically upregulated by TGF-b in intratumoral CD4+ T cells (35%). In addition, lymphoma B cells strongly enhanced Foxp3 expression in intratumoral CD4+CD25−Foxp3−. Furthermore, when added together, the frequency of Foxp3+ T cells and Foxp3-inducible cells reached up to 60% of CD4+ T cells in tumor microenvironment of B-cell NHL. These findings suggest that the balance of effector TH17 cells and inhibitory Treg cells is disrupted in B-cell NHL and significantly favors the development of inhibitory Treg cells. Our data indicate that lymphoma B cells are key factor in regulating differentiation of intratumoral CD4+ T cells toward inhibitory CD4+ T cells.

Abstract Background The role of intratumoral PD-1+ T cells and their contribution to patient outcome in follicular lymphoma (FL) has been controversial. While some studies have found that increased numbers of PD-1+ T cells correlate with an inferior prognosis or have no impact on patient outcomes, other studies observed that the numbers of intratumoral PD-1+ T cells are an indicator of a favorable outcome in FL. In previous work, we observed that PD-1 can be expressed by different subsets of intratumoral T cells in FL including T follicular helper (TFH) cells and exhausted T effector cells. However, the function, prevalence, distribution and clinical importance of the respective intratumoral PD-1+ T subsets in FL patients are largely unknown. Goal To characterize PD-1+ T cell subsets and to determine the biological and clinical relevance of these T cell subsets in FL. Results In an analysis of biopsy specimens from 33 previously untreated FL patients, we found that PD-1 is abundantly expressed on intratumoral T cells. A median of 54.9% (range: 17.2-92.6, n=33) of CD4+ or 45.8% (range: 12.8-81.7, n=33) of CD8+ T cells express PD-1 on the cell surface. We found by flow cytometry that PD-1 is expressed on intratumoral CD4+ T cells with bright or dim intensity that represents two different subpopulations in FL. By immunohistochemistry, we identified that CD4+PD-1bright T cells predominantly reside in the lymph node follicles while PD-1dim T cells are mainly located in an inter-follicular pattern. Intratumoral CD4+PD-1bright T cells have a TFH cell phenotype and express CXCR5, secret IL-21 and are BCL-6 positive with no TIM-3 expression. In contrast, CD4+PD-1dim T cells have an exhausted phenotype, express TIM-3 and do not express BCL-6 and CXCR5. Functionally, CD4+PD-1dim T cells displayed reduced proliferation, cytokine production and cell signal transduction. When the CD8+ T cells were analyzed, we found that intratumoral CD8+ T cells, primarily reside in the inter-follicular regions, express PD-1 with low intensity and exhibit similar phenotypic and functional changes to inter-follicular CD4+PD-1dim T cells. These cells express TIM-3 but lack CXCR5 and BCL-6. They also displayed reduced proliferation and cytokine production. Clinically, we observed that both the numbers of CD4+PD-1dim and CD8+PD-1dim exhausted T cells significantly correlate with a reduced progression-free survival in FL patients treated with single agent rituximab (p=0.007 and p=0.04 respectively; n=31). Although it did not reach statistical significance, CD4+PD-1bright TFH cells appeared to correlate with a favorable survival, suggesting that TFH and exhausted T cells differentially impact patient outcome in FL. Conclusion Taken together, these results indicated that PD-1 expression defines two subpopulations with distinct functions that differentially impact patient outcome in FL. This finding provides not only an explanation for the previously discrepant clinical observations, but is also important in the design of future anti-PD-1 antibody-based therapy in FL. Disclosures: No relevant conflicts of interest to declare.

Abstract Gene expression profiling has shown that diffuse large B-cell lymphoma (DLBCL) clusters into three major subtypes based on similarity in expression patterns to their cell of origin (COO): germinal center B-cell-like (GCB-DLBCL), activated B-cell-like DLBCL (ABC-DBLCL) and primary mediastinal B-cell lymphoma (PMBCL). These subtypes of DLBCLs are associated with distinctly different overall survival rates after standard immunochemotherapy. However, clinical and prognostic heterogeneity remains within COO subsets and strategies are needed to further stratify patients to identify and target high-risk subsets. A comprehensive genomic analysis of COO on a clinically defined set of DLBCL cases has not been performed and the aim of this study was to use whole-exome sequencing (WES) data from 58 paired tumor-normal DLBCL samples to assess association of known DLBCL genomic alterations with cell COO as well as for identification of novel and relevant genetic biomarkers. To investigate genomic alterations associated with DLBCL subsets, we analyzed WES and genome wide copy number data from 58 paired tumor-normal DLBCL tumors. Gene expression profiling or Hans classification was performed to determine DLBCL COO subtype; 31 patients were classified as GCB and 27 as non-GCB. The WES data were used to 1) assess the association of known DLBCL genomic alterations with COO, and 2) identify novel alternations associated with COO. Statistical analysis was performed and the data were ranked by significance (p≤ 0.05) within each DLBCL subtype. In total, 45 genomic abnormalities were analyzed for their association with either GCB, non-GCB or both. Mutations in CREBBP, EZH2, MEF2B, FOXO1 and REL have also been reported as GCB driver mutations and we observed GCB patients with these mutations, but the mutation clustering was not wholly associated with GCB. MYC double-hits were exclusively found in the GCB-subtype group. For the non-GCB cases we found that mutations in MAP2K3 and MYD88 were significantly associated with this subtype(p< 0.05). In addition to mutational patterns, we identified several copy number alterations (CNA) across both groups. Chromosomal losses in GCB patients were found at chromosomes 10q11.21-10q24.23, 4q12-4q35.2, 3q12.1-3q29, 4p12-4p16.3, 10p11.21-10p15.3, and 14q11.2-14q24.3 whereas gains were localized to 7q11.1-7q36.3, 7p11.2-7p22.3, and 1q21-1q32.1 (p < 0.05). No CNA was observed to directly associate with non-GCB patients, however, a loss at 9p21 and gains at 9q24.1 and 18q21.33 trended with the non-GCB subtype, supporting previous reports. Loss at 10q23.31 or a gain in 2p13-2p12 have been reported as being specific for GCB and our data confirmed the association of 10q23.31 with GCB while a gain at 2p13-2p12 (REL)was found in both subtypes. To further understand genomic differences between DLBCL subtypes, we evaluated the relative percentage of each genomic feature. 18/45 (40%) were only observed in GCB patients whereas 2/45 (5%) were specific to the non-GCB subtype. The majority (25/45, 55%) overlapped between the two subtypes. Throughout our analysis we noted that 7 non-GCB cases lacked any of the driver mutations analyzed in the study. While all cases carried mutations, they consisted of low frequency mutations that were not specifically associated with COO. 2/7 cases had a gain at 9p24.1 that included CD274 and JAK2. Because 9p24.1 gains have not been fully defined in DLBCL, we reviewed all cases and identified 4 (7%) with a 9p24.1 gain, 3 of which were non-GCB and 1 GCB. One non-GCB case was EBV+ and none of the cases showed evidence of PMBCL. Of the 9p24.1 cases, three had RNAseq data available and we found that PDL1 and JAK2 expression was elevated (12 fold p< 0.01 and 7 fold p< 0.01, respectively) when compared to the 9p24.1 normal cases (n=32). While outcome was not the focus of the study, we did note that 6/7 cases that lacked driver mutations achieved event free survival at 24 months (EFS24). Taken together, this analysis has further characterized the genetic profile of each COO subtype and has identified novel GCB CNAs which require independent replication. Additionally, we identify a subgroup of non-GCB DLBCL patients that do not harbor known driver mutations and require further genomic study to better resolve the biology of these tumors. Together, these data provide insight on the genetic heterogeneity of DLBCL and identify genetic variants that may inform subtype specific therapy. Disclosures Nowakowski: Morphosys: Research Funding; Celgene: Research Funding; Bayer: Consultancy, Research Funding. Rimsza:NCI/NIH: Patents & Royalties: L.M. Rimsza is a co-inventor on a provisional patent, owned by the NCI of the NIH, using Nanostring technology for determining cell of origin in DLBCL..