Novak, Anne J.

Abstract Diffuse large B-cell lymphoma (DLBCL) is clinically and biologically heterogenous. Based on gene expression profiling of the tumor, DLBCL is classified into two major molecular subtypes linked to cell-of-origin (COO): germinal center (GCB) and activated B-Cell (ABC). The GCB subtype is characterized by a high prevalence of somatic mutations, particularly in apoptosis genes (e.g., BCL2) which is characteristic of follicular lymphoma (FL, another GCB lymphoma). The development of the validated NanoString gene expression test as well as immunohistochemistry markers that work in formalin-fixed, paraffin-embedded tissue allows for the classification of DLBCL by COO in epidemiological studies. We hypothesized that integration of COO with germline genetics could provide insight into the underlying biology driving this heterogeneity, as well as insight into putative function of germline risk variants. Prior genome wide association studies (GWAS) have identified 7 SNPs in 6 susceptibility loci for DLBCL, including: 2p23.3 (rs79480871 NCOA1), 3q13.33 (rs9831894 CD86), 3p24.1 (rs6773363 EOMES), 6p21.33 (rs2523607 HLA-B), 6p25.3 (rs116446171 EXOC2), and 8q24.21 (rs13255292 and rs4733601 PVT1 and MYC). Two of these loci were also identified in FL GWAS (different lead SNPs): 6p21.33 (rs3130437) and 8q24.21 (rs13254990 PVT1). Additional published FL susceptibility loci include: 3q28 (rs6444305 LPP), 6p21.32 (rs9268839), 11q23.3 (rs4938573 CXCR5), 11q24.3 (rs4937362 ETS1), and 18q21.33 (rs17749561 BCL2). Here we evaluated whether the published DLBCL/FL loci differ by COO molecular subtypes. We evaluated each of the 14 DLBCL/FL SNPs by case-case (GCB vs. nonGCB DLBCL) and polytomous case-control (GCB, nonGCB, and FL vs. controls) analyses. DLBCL cases were classified into GCB vs. nonGCB based on best available data from NanoString, RNAseq, followed by the Hans algorithm. A total of 778 DLBCL cases (470 GCB and 308 nonGCB), 1,050 FL cases, and 1,383 controls were analyzed. Odds ratios (OR) based on ordinal encoding of each SNP and 95% CI were calculated for each of the comparison groups. The ORs for four GWAS SNPs were significantly elevated in GCB vs. nonGCB DLBCL as well as in FL; two were FL GWAS SNPs from the HLA region (6p21.32 and 6p21.33) and the other two (BCL2 and ETS1) are important in the germinal center program. In contrast, one SNP (EXOC2) was significantly elevated in nonGCB-DLBCL and was not associated with GCB-DLBCL or FL; this SNP is also near IRF4 (MUM1), a gene important in the ABC program. For the remaining SNPs, there were suggestive associations for GCB-DLBCL and FL versus nonGCB DLBCL (CD86, CXCR5); nonGCB-DLBCL vs. GCB-DLBCL and FL (NCOA1, PVT1); and associations that showed no pattern with COO (EOMES, LPP, HLA-B and MYC). These findings provide insight into the shared pathogenesis of FL and GCB-DLBCL to inform etiologic mechanisms and risk assessment. Citation Format: Rosalie Griffin Waller, Dennis P. Robinson, Anne J. Novak, Lisa M. Rimsza, Kerstin Wenzl, Rebecca L. King, Andrew L. Feldman, Matthew J. Maurer, Grzegorz S. Nowakowski, Brian K. Link, Thomas M. Habermann, Susan L. Slager, James R. Cerhan. Etiologic heterogeneity of genetic risk for DLBCL cell-of origin molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1181.

Abstract The tumor microenvironment (TME) plays a crucial role in mediating the tumor immune response, thereby affecting patient outcomes in follicular lymphoma (FL). Using CyTOF, we analyzed a cohort of 82 FL patients with biopsy specimens collected before the first treatment regimen was administered. Our results showed that there was a difference in the T-cell phenotype in patients with various TMEs. Patients with TMEs rich in monocyte/macrophages/NK cells had more CD8+ cells and tended to have fewer TN, but more TEMRA and TEXH cells. Patients enriched with T cells exhibited a T-cell phenotype skewed toward to TCM and patients with B cell dominance tended to have significant higher number of TFH cells. T-cell phenotypes with more terminally differentiated, senescent and exhausted cells were highly represented in patients with less T cells when compared to patients with more T cells. We observed that CD57+ TFH cells was significantly more abundant in patients who had disease progression than patients who had complete response to therapy, suggesting a role for CD57+ TFH cells in promoting malignant cell growth in FL. Consistent with this finding, we observed that increased numbers of CD57+ TFH cells correlated with an inferior survival in FL. Using CITE-seq technology, we found that CD57+ TFH cells exhibited a substantially different transcriptome from CD57− TFH cells. Genes that were differentially upregulated in CD57+ TFH cells when compared to CD57− TFH cells included genes involved in cell survival, compromised inflammatory response, and metabolic activation. Taken together, our results indicate different tumor microenvironments among patient groups that is associated with variable T-cell phenotypes.

Abstract T cell Ig and ITIM domain (TIGIT) is an immune checkpoint molecule and its ligation delivers an inhibitory signal to T cells that negatively regulates anti-tumor responses. However, the expression and biological relevance of TIGIT in B-cell non-Hodgkin lymphoma (NHL) is completely unknown. To phenotypically characterize TIGIT+ T cell subsets, we profiled TIGIT-expressing CD4+ and CD8+ T cells from biopsy specimens of B-cell NHL. TIGIT expression was not detectable on T cells from peripheral blood, however, TIGIT is constitutively expressed on CD4+ T cells from biopsy specimens of B-cell NHL with a median of 44.8% (range: 25.9–62.2, n=8) of CD4+ T cells expressing TIGIT. By profiling the CD4+TIGIT+ T cells, we observed that CD25 was highly expressed on intratumoral CD4+TIGIT+ T cells. Intracellular staining revealed that Foxp3 was expressed by intratumoral CD4+TIGIT+ T cells. These intratumoral CD4+TIGIT+ T cells expressed high level of chemokine receptor CCR4 and were absent of IL-7 receptor-α (CD127). Functionally, CD4+TIGIT+ T cells displayed reduced cytokine production, as the number of IFN-γ- and TNF-α-producing cells was lower in the TIGIT+ population than in TIGIT− T cells. While TIGIT expression can be induced by TCR activation, treatment of T cells with IL-2 and TGF-β inhibited TCR-mediated TIGIT induction. Furthermore, lymphoma B cells were involved in TIGIT induction as in vitro depletion of lymphoma B cells altered upregulation of TIGIT on CD4+ T cells in B-cell NHL. Taken together, these results indicated that constitutive expression of TIGIT defines a CD4+ population with similar phenotype to regulatory T cells. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression in B-cell NHL

Abstract Although extensively studied, the exact mechanism by which TGF-β mediates T-cell inhibition is not entirely clear. When we examined the effect of TGF-β on the phenotype and function of T cells, we noted that CD70 was significantly up-regulated by TGF-β. This surprising finding led us to explore the underlying mechanism by which TGF-β upregulates CD70 expression on T cells and to define the function of CD70+ cells in B-cell NHL. Interestingly, we observed that TGF-β preferentially induced CD70 expression on effector memory T cells while it predominantly up-regulated Foxp3 on naïve T cells. CD70 induction is Smad3-dependent and involves Stat5 signaling. CD70+ T cells were highly susceptible to TGF-β-mediated apoptosis and that transfection of Jurkat cells with the CD70 gene led to a significantly reduced survival. TGF-β-induced CD70+ T cells had a diminished response to immune stimulation associated with downregulation of co-stimulatory molecules CD27 and CD28. CD70-expressing T cells from biopsy specimens of patients with B-cell NHL failed to produce cytokines and displayed no signal transduction when stimulated. CD70-expressing T cells were highly represented in follicular NHL biopsy specimens and increased numbers of CD70+ cells correlated with an inferior patient outcome. These novel findings not only revealed a biological role for a TGF-β/CD70 axis in mediating immune inhibition, but also may lead to therapeutic approaches in patients with B-cell NHL.

Abstract Purpose: IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences. Experimental Design: We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry. Results: We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism. Conclusions: We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications.