Citations

Abstract Background: Although CAR T-cell therapy has achieved remarkable response rates in adult patients (pts) with relapsed and refractory B-cell non-Hodgkin’s lymphoma (NHL), 20-30% of pts still have primary refractory disease, and 30-40% of pts relapse, often within 6 months of therapy. Given that durable clinical response can be achieved without CAR T cell persistence, we hypothesized that durable remission is correlated with the expansion of tumor-reactive T-cell receptor (TCR) clones following CAR T-cell infusion. To assess this, we examined the TCR repertoire of adult pts with B-NHL who received CD19 CAR T-cell (axi-cel) therapy to identify correlates of clinical response. Methods: Single-cell (sc) RNA sequencing (seq) and scTCR-seq were performed on peripheral blood mononuclear cells from B-NHL pts before lymphodepletion chemotherapy (pre), at peak CAR-T expansion (peak), and one month following CAR-T (M1). scRNAseq and scTCR-seq data were processed using CellRanger v7.0.1, which were then analyzed using Immunarch (v0.9.0) for TCR clonality and sample diversity. Diversity was assessed using the Gini coefficient to indicate clone size inequality (0 = maximum diversity). TCR diversity and phenotype of expanded and emerged TCR clones post CAR-T were compared across clinical response categories: CR (complete remission for at least 6 months), PD1 (primary refractory), and PD2 (relapsed after partial or complete response). Results: We analyzed 107,035 T cells from 77 serial samples of 32 pts (16 CR, 4 PD1, 12 PD2) and 5 healthy controls. Compared to pts with PD1 and PD2, pts with CR had a larger Gini coefficient for TCR clones at M1 relative to Pre and Peak. The Gini coefficient was the largest for tumor recirculating CD8 T effector memory (Tem) and activated CD8 peripheral memory (Tpm) cells at M1 for pts with CR. Examining the repertoire of emerged and expanded TCR clones at Peak and M1 from Pre, pts with PD2, compared to pts with CR, had a higher percentage of activated CD8 T peripheral memory (Tpm) and T regulatory cells (Treg) among the emerged clones and activated CD8 Tpm among the expanded clones. Patients with PD1, relative to patients with CR, had more proliferating and non-activated CD8 Tpm among the expanded and emerged clones. Conclusion: NHL pts with CR after CAR-T had more robust clonal expansion of T cells, particularly tumor recirculating CD8 Tem and activated CD8 Tpm, compared to pts with relapsed or refractory disease. In contrast, pts with relapsed or refractory disease had an expansion of TCR clones with a larger proportion of CD8 Tpm and Tregs. Endogenous tumor-reactive TCR expansion may help predict favorable clinical response following CAR T-cell therapy. Citation Format: Aiman J. Faruqi, Panwen Wang, Radhika Bansal, Andre De Menezes Silva Corraes, Henan Zhang, Zuoyi Shao, Kevin Regan, Eider Moreno Cortes, Arushi Khurana, Nora Bennani, Yucai Wang, Paul Hampel, Jonas Paludo, Patrick Johnston, Hemant Murthy, Madiha Iqbal, Stephen Ansell, Javier Munoz, Allison Rosenthal, Mohamed Kharfan Dabaja, Januario E. Castro, Saad Kenderian, Haidong Dong, Ying Li, Yi Lin. T-cell receptor repertoire changes associated with clinical response in patients with B-cell non-Hodgkin’s lymphoma receiving CD19 CAR-T therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 53.

Abstract Despite the remarkable efficacy of chimeric antigen receptor T -cell therapy (CART) for the treatment of B-cell malignancies, durable remission remains limited often due to tumor relapse. We have previously demonstrated that tumor-derived extracellular vesicles (EVs) induced CART cell failure in preclinical models. To characterize the impact of tumor EV induced CART cell dysfunction, we assessed microRNA (miRNA) profiles contained within tumor EVs from two independent patient cohorts and studied how they contributed to CART dysfunction. Our first cohort included patients with chronic lymphocytic leukemia (n=50). We previously showed that CLL patient derived EVs induce a state of CART cell dysfunction. To understand this dysfunction, we interrogated the miRNA cargo significantly enriched in CLL EVs and how their coculture impacts CART signaling by RNAseq. Here, we found a significant enrichment in miR-125a within CLL EVs as compared to healthy donor controls (p=.0001). CART cocultured with CLL EVs showed alterations in T cell activation pathways, where miR-125a targets TNFAIP3 and NFkB pathways were activated. These findings implicate miR-125a from CLL EVs in CART dysfunction. Next, we aimed to validate the impact of miRNA on CART cell outcomes in the clinic. We interrogated miRNA profiles from patients with diffuse large B cell lymphoma (DLBCL) treated with CART cell therapy (CART19-28ζ, axicabtagene ciloleucel). EV miRNA cargo was compared between responders (complete remission at 6 months, n=10) and non-responders (no response 1 month post CART19, n=10) to identify differentially enriched miRNAs across groups. miR-125a was again significantly upregulated in EVs of non-responders as compared to responders to CART19 treatment (fold change=3.28, padj=.018). To study the direct effect of miR-125a on CART cell functions, we generated and studied the effector functions of CART19-28z following transfection with miR-125a or negative miR control. CART19 cells transfected with miR-125a showed a dose dependent decrease in killing of lymphoma cells as compared to miR-control (p=.0001). miR-125a also significantly increased the rate of apoptosis in CART19 cells (fold change=1.95, p=.024) and decreased the level of CART proliferation (fold change=.71, p=.013) as compared to controls. Notably, miR-125a did not impact tumor cell growth, further verifying a direct CART effect. In summary, our studies demonstrate the impact of tumor derived miRNAs on CART cell outcomes. They identify that miR-125a is 1) associated with CLL EV induced CART dysfunction, 2) significantly elevated in non-responders to CART therapy in the clinic, and 3) able to decrease CART19 efficacy through reduced CART killing/proliferation and increased apoptosis. These results highlight a novel mechanism of CART19 failure induced through miRNA cargo delivery by tumor-derived EVs and the role of miR-125a in inducing CART19 dysfunction. Citation Format: Olivia Sirpilla, R. Leo Sakemura, Ismail Can, Truc N. Huynh, James H. GIrsch, Claudia Manriquez Roman, Carli M. Stewart, Kun Yun, Ekene J. Ogbodo, Long Mai, Omar Gutierrez Ruiz, Makena Rodriguez, Brooke Kimball, Hong Xia, Jose C. Villasboas, Stephen Ansell, Nora Bennani, Patrick B. Johnston, Jonas Paludo, Januario Castro, Mohamed Kharfandabaja, Sameer Parikh, Neil Kay, Yi Lin, Elizabeth Siegler, Saad Kenderian. Tumor-derived microRNA125a induces CART cell apoptosis and failure in B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3841.

Abstract Immune checkpoint inhibitors (ICIs) have demonstrated remarkable response rates in relapsed or refractory Hodgkin lymphoma (HL). Still, most patients eventually progress. Patterns of progression after ICIs are not well described and are essential to defining the role of local therapies in combination with ICIs. We identified patients who received ICIs for HL between 2013 and 2022. Fludeoxyglucose-18 positron emission tomography (FDG-PET) before initiating ICI and at progression on/after ICI were reviewed, and areas of active HL were recorded. An exploratory analysis of treatable progression included patients with ≤5 sites of disease on pre-ICI FDG-PET and progression only at pre-ICI sites. Ninety patients were identified; 69 had complete records, and of these, 32 (52%) had relapsed at ICI initiation, 17 (25%) were refractory, and 16 (23%) received ICI as first-line therapy. Forty-five of 69 patients had ≤5 sites of disease (limited) on pre-ICI FDG-PET. Patients with >5 sites of disease had a higher risk of progression, and every site of disease >5 sites conferred an additional 1.2x higher chance of progression. At a median follow-up of 4.0 years, 41 of 69 patients had progressed on/after ICIs (cumulative incidence 66.4%), and of these, 22 of 41 patients progressed only at pre-ICI sites (cumulative incidence 39.4%). In an exploratory analysis, the cumulative incidence of a treatable progression among 45 patients with limited disease was 34%. The cumulative incidence of any progression among this cohort was 58.9%. More than one-third of patients with limited disease before ICIs experienced progression only at pre-ICI sites of disease. These patients could be candidates for radiation during or after ICIs.

Background:Waldenström Macroglobulinemia (WM) is a rare, indolent B‐cell lymphoplasmacytic malignancy. Patient outcomes are highly heterogeneous, with a proportion of patients surviving less than 5 years (short survivors) and another proportion that survive longer than 10 years. The laboratory parameters in the currently used International Prognostic Scoring System for WM (IPSSWM) include hemoglobin, serum IgM, platelet count, and serum β2‐microglobulin.Aims:We aim to identify unique patient characteristics and prognostic features associated with short survival in WM.Methods:The medical records of patients with active WM that were diagnosed at Mayo Clinic Rochester, Arizona, and Florida between January 1st, 1996 and December 31st, 2013 were reviewed. The patients with smoldering WM were excluded. Cohorts of patients who either survived ≤ 5 years (Short Survival Group) or those with an overall survival (OS) ≥ 10 years (Long Survival Group) from the diagnosis of active disease were compared. Survivors with a follow‐up of ≤ 5 years were not included in the analyses. Two‐sided Wilcoxon rank sum test and Chi square/Fisher's exact test were used to compare the continuous and categorical variables, respectively. The variables that were statistically significant on univariate analysis (P < 0.05) were included in a multivariate logisitic regression analysis.Results:Of 893 consecutively seen WM patients (median follow‐up of 9 years [95% CI 8.4–9.7]), we identified 387 patients who could be categorized into either the Short Survival Group (n = 171 patients, 44%) or the Long Survival Group (n = 216, 56%). The Short Survival Group had a median OS of 2.4 years (2.1–2.8) and median age at diagnosis of 73 years (42–93), while the Long Survival Group (n = 216, 56%) had a median OS of 18.9 years (16‐NR) and median age at diagnosis of 62 years (31–85). The baseline characteristics and significant findings between the two groups are listed in the Table. Parameters significant on univariate analysis included age >65 years, albumin < 3.5 g/dL, β2‐microglobulin >3 μg/ml, and LDH > upper limit of normal (ULN). On multivariate analysis, albumin <3.5 g/dL and LDH > ULN were independently associated with short survival. Among the Long Survivors, 60 (28%) patients survived 15 years or more.Summary/Conclusion:Lactate dehydrogenase above the ULN and serum albumin <3.5 g/dL are independent biomarkers of short survival in WM. The MYD88 L265P status and the size of serum IgM are not prognostic in WM. Our data suggest the need for simplification of the current staging system with the use of widely available markers: serum albumin and lactate dehydrogenase.image

Background:Ibrutinib is approved in Europe and the US for the treatment of Waldenstrom macroglobulinemia (WM) with a median progression‐free survival (PFS) measured on several years. As PFS is commonly used for drug approval by regulatory entities, there is an unmet need of reliable surrogate markers for PFS that could help accelerating such process. This is of importance with novel BTK inhibitors currently in clinical trials.Aims:We hypothesized that the depth of response at 6 months can be predictive of PFS in patients with WM treated with the BTK inhibitor ibrutinib.Methods:We evaluated our hypothesis in two separate cohorts of WM patients treated with ibrutinib. The learning cohort was composed by participants of two prospective clinical trials at Dana‐Farber Cancer Institute (DFCI), Memorial Sloan Kettering Cancer Center and Stanford Cancer Center. The validation cohort was composed by consecutive WM patients treated with ibrutinib off clinical trial at DFCI and Mayo Clinic. Categorical responses at 6 months were assessed based on criteria from the 6thInternational Workshop for WM. PFS6 was defined as the time from the 6‐month response assessment until progression of disease or death from any cause. PFS6 curves were generated using the Kaplan‐Meier method and compared using the log‐rank test. Cox proportional hazard regression models were fitted for PFS6. Pvalues <0.05 were considered statistically significant.Results:The learning and validation cohorts were composed by 92 and 190 WM patients treated with ibrutinib, repsectively. There were no statistical differences between cohorts with regards to age, sex, serum IgM level, hemoglobin level, bone marrow involvement, prior treatment status, and MYD88 and CXCR4 mutational status (p>0.05 for all variables). The rates of partial response (PR) or better at 6 months in the learning and validation cohorts were 63% (54/86) and 72% (114/159), respectively (p = 0.15). The median PFS6 was not reached in the learning cohort and was 53 months (95% CI 44‐not reached) in the validation cohort. In the learning cohort, the median PFS6 for patients who attained a PR or better at 6 months was not reached, while for the patients who did not attained PR at 6 months was 48 months (95% CI 33‐not reached). The 3‐year PFS rate for patients who attained a PR or better at 6 months was 83% (95% CI 68–91%), while for patients who did not attained PR at 6 months was 58% (95% CI 33–76%) (Figure, left, log‐rank p = 0.02). Attaining PR or better at 6 months was associated with a hazard ratio (HR) or progressing or dying of 0.37 (95% CI 0.16–0.87; p = 0.02).In the validation cohort, the median PFS6 for patients who attained a PR or better at 6 months was 53 months (95% CI 44‐not reached), while for the patients who did not attained PR at 6 months was 32 months (95% CI 22‐not reached). The 3‐year PFS rate for patients who attained a PR or better at 6 months was 79% (95% CI 67–87%), while for patients who did not attained PR at 6 months was 50% (95% CI 26–70%) (Figure, right, log‐rank p = 0.04). Attaining PR or better at 6 months was associated with a hazard ratio (HR) or progressing or dying of 0.46 (95% CI 0.22–0.98; p = 0.04). Age, sex, serum IgM level, hemoglobin level, bone marrow involvement and prior treatment status were not prognostic of PFS6 in both cohorts.Summary/Conclusion:Attaining a PR or better at 6 months to ibrutinib is associated with longer median PFS and higher rates of 3‐year PFS in WM patients. Depth of response at 6 months could serve as a surrogate for PFS in clinical trials evaluating BTK inhibitors in WM patients.image

AbstractIntroductionThe infusion of autograft Natural Killer Cells (NKC)/CD14+HLA‐DRDIM ratio is a predictor of survival in lymphoma patients undergoing autologous peripheral blood hematopoietic stem cell transplantation (APBHSCT). This study evaluated if the Day 100 NKC/CD14+HLA‐DRDIM ratio still functions as a prognostic immune‐biomarker.MethodsThis was a retrospective, single‐institution, cohort analysis including 107 patients in this study that had clinical assessment at Day 100 post‐APBHSCT from our prior phase III trial. We evaluated the prognostic ability of the Day 100 NKC/CD14+HLA‐DRDIM ratio to predict overall survival (OS) and progression‐free survival (PFS) using Cox regression model for outcome analysis and survival by Kaplan–Meier method.ResultsThe median follow‐up from day 100 was 94.7 months (range 4.83–158.1 months) for the entire cohort. Patients with a Day 100 NKC/CD14+HLA‐DRDIM ratio ≥1.67 experienced better OS and PFS versus those with a Day 100 NKC/CD14+HLA‐DRDIM ratio <1.67: median OS was not reached versus 49.7 months, the 5‐year OS rates were 91% (95% CI, 81%–96%) versus 40% (95% CI, 27%–55%), p < .0001, respectively; and median PFS was not reached versus 23.5 months, the 5‐year PFS rates were 66% (95% CI, 55%–81%) versus 21% (95% CI, 15%–40%), p < .0001, respectively. Day 100 NKC/CD14+HLA‐DRDIM ratio was an independent predictor for OS and PFS in the multivariate analysis.ConclusionsDay 100 NKC/CD14+HLA‐DRDIM ratio is a prognostic immune‐biomarker in lymphoma patients post‐ APBHSCT.

Introduction: In Cohorts 1+2 of the ZUMA-1 Phase 1/2 study of axi-cel in R/R LBCL, 11% and 31% of patients (pts) experienced Grade ≥3 cytokine release syndrome (CRS) or neurologic evenets), respectively. To minimize the toxicities seen with axi-cel, a safety management cohort, Cohort 6 (N=40), was added to evaluate whether prophylactic (ppx) corticosteroids (CS) could decrease grade ≥ 3 CRS or ICANS. The 2-year update of ZUMA-1 Cohort 6 showed no grade ≥3 CRS and 15% grade ≥3 ICANS without any difference in objective response rate (ORR) or complete response rate (CR) when compared to cohorts 1+2 (Oluwole Blood 2022). The FDA had previously approved an update to the prescribing information for axi-cel to include use of ppx CS across all approved indications in January 2022. However, ppx CS is not a mandatory standard treatment and we sought to establish the clinical outcomes in the real-world practice. Methods: To assess the clinical outcomes of pts receiving ppx CS, we conducted a multicenter retrospective analysis of pts with R/R LBCL who were treated with axi-cel since the FDA approval of ppx CS in January 2022. Baseline characteristics, toxicities, and efficacy were compared between the no ppx and ppx CS groups. CRS and ICANS were graded based on ASTCT 2019 guidelines. Event-free survival (EFS) was defined as time from axi-cel infusion to progression, re-treatment, or death. Overall survival (OS) was defined as time from axi-cel infusion to death due to any cause. Results: A total of 66 pts at 5 centers received axi-cel between Jan 31, 2022, and June 20, 2023. Thirty-five (52%) pts received ppx CS, with a median age of 63 years (26-85 years). By histology, 75% (n=50) of the pts had DLBCL, 13.6% (n=9) had high grade B-cell lymphoma (HGBL), 9.1% (n=6) had transformed follicular lymphoma (TFL) and 1.5% (n=1) primary mediastinal LBCL (PMBCL). 59% of the pts (n=37) had IPI &gt;2 and 66% (n=43) had stage III/IV disease. The baseline characteristics at the time of apheresis are shown in Table 1 and were comparable between the 2 groups, except for more HGBL, fewer prior lines of therapy and longer median time from apheresis to axi-cel infusion in the ppx CS group. Among the entire cohort, only 37% (n=25) met ZUMA-1 eligibility criteria at the time of lymphodepletion, and 67% (n=44) needed bridging therapy. Any grade CRS occurred in 88% (n=58) of the pts, with grade ≥2 CRS in 38% (n=25). Any grade ICANS occurred in 55% (n=37) of the pts, with grade ≥2 ICANS in 41% (n=27). Any grade ICANS was significantly lower in the ppx CS group than the no ppx group (45.7% vs 65.5%). The rates of any grade CRS, grade ≥2 CRS, grade ≥2 ICANS, the median time to CRS onset, ICANS onset, CRS duration, and ICANS duration were comparable between the 2 groups. Tocilizumab and steroids use was similar between two groups with a trend towards less ICU admission in patients in the ppx CS group. Overall, 14 (21%) pts developed infectious complications within 30 days of axi-cel infusion. The day 30 ORR was 84% and was comparable between the 2 groups (77% in the ppx group vs 93% in the no ppx group). The CR was 45.7% in ppx CS group compared to 65.5% in no ppx group. At a median follow up of 219 days (27-1178), 37 (56.1%) pts had progressed and 25 (37.9%) pts had died. The number of days to progress was significantly lower in the ppx CS arm. The median EFS and OS were not significant between the 2 groups. Conclusions: This multicenter retrospective study showed that the efficacy of treatment with axi-cel in R/R LBCL did not decrease with the use of ppx CS. No significant differences in efficacy or CAR-T-associated toxicities were noted in the 2 groups, potentially due to selective use with patients at presumed higher risk of severe CRS and ICANS. A larger dataset and longer follow-up is needed for risk-stratified analysis and the study is ongoing.

Despite notable advancements in treating chronic lymphocytic leukemia (CLL) with targeted therapies, a significant clinical challenge arises as a subset of CLL patients progress and develop Richter's transformation (RT), an aggressive form of lymphoma. While immune cell dysfunction and T cell defects are well-known characteristics of CLL, the alterations in immune cell subsets during CLL progression to RT remain largely unexplored. While CLL patients typically fail to respond to anti-PD1 pembrolizumab, a positive response has been observed in 20-40% of RT patients. Previously, our study involving three RT patients (1 responder and 2 progressors) revealed enriched regulatory T cells (Tregs) in the responders compared to progressors after pembrolizumab treatment (Iyer et al, ASH2020). Hence, we propose that dynamic changes in T cell subsets at baseline may be associated with response to pembrolizumab treatment. To this end, we performed single-cell RNA sequencing on sorted T cells (CD3 +) from PBMCs derived from 20 patients (responders (RES), n=5; stable disease (SD), n=5; progressive disease/progressors (PD), n=10). A total of 408,627 cells were captured, and an average of 1,393 genes were detected per cell per patient. Using a graph-based clustering approach (Seurat V4.3), we identified 19 clusters based on marker genes and transcriptional signatures and identified enriched pathways using Enrichr analysis. Our results showed that RES had an increased Th17 subset at baseline compared to PD (15% vs. 5%, p=0.01) and reduced Th2 T cells compared to SD (p=0.004) and PD (p=0.05). The Th17/Th2 ratio was higher in RES at baseline (Th17/Th2 R: 1.24, PD: 0.21, SD: 0.25, p0.05) and post-pembrolizumab (Th17/Th2 RES: 1.31, PD: 0.34, SD: 0.30, p0.05). Furthermore, RES had an increased fraction of proliferating CD4 T cells at baseline (4% RES vs. 0.4% SD and 0.4% PD, p=0.02) and post-treatment (1.9% RES vs. 0.8% PD and 0.34% SD, p=0.02). Although we observed an increasing trend in the fraction of Tregs in the RES at baseline [(7.6% RES vs.6.1% PD and 6.5% SD, p=0.4; ns) and post-treatment (10.3% RES vs. 8.3% PD and 5.8% SD, p= 0.2; ns] compared to PD and SD, the difference was not significant. Similarly, among CD8+ T cells, we observed an increasing trend in CD8+ effector T cell fraction in the responders at baseline (29.8% RES vs. 21% PD and 15.6% SD, p=0.9, ns) and post-treatment (30.1% RES vs. 21.2% PD and 19.9% SD, p=0.44, ns). However, the difference was not statistically significant. These findings highlight the dynamic changes in T cell subsets associated with pembrolizumab response. In terms of transcriptional changes in Tregs following pembrolizumab treatment among RES, PD, and SD, we observed dynamic changes on a few Treg marker genes, including TIGIT and TOX. TIGIT and TOX expression drastically decreased after treatment in RES (p &lt; 0.0001), while expression did not change in PD and SD. There were no significant changes in the expression of FOXP3 or CD27 in RES, PD, and SD before and after the treatment. In CD8 effector T cells, GZMK, GZMA (granzymes), and PRF1 (perforin) are marker genes. We observed higher expression of GZMK and GZMA in RES (p &lt;0.0001) at the baseline compared to SD and PD. In the proliferating CD4 T cells, we observed increased expression of IDH2 (Isocitrate dehydrogenase 2-key enzyme of TCA cycle) in RES at the baseline, as compared to PD and SD, indicating increased TCA cycle activity in the proliferating T cells. Pathway analysis of T cell clusters using the Enrichr database showed TNFA-IFNG-MYC targets are enriched in CD8 + cells, IL2-STAT5 signaling is upregulated in Th17, TREGs, and E2F cell cycle associated pathways enriched in CD4 + proliferation cluster. In conclusion, our study unveils significant baseline differences in CD4 subsets between RES and PD/SD subjects and dynamic changes in T cell subsets and transcriptional signatures. These findings offer potential predictors of pembrolizumab response in RT, such as increased Th17/Th2 ratio, enriched proliferating CD4 + T cells, and elevated TIGIT, TOX gene expression in Tregs, GZMK, and GZMA in CD8 effector cells. This deeper understanding of the immunological landscape in RT patients sheds light on the interplay between T cell subsets and treatment response in RT, paving the way for personalized therapeutic strategies. Further validation and exploration can lead to improved outcomes and tailored interventions for RT.

Introduction: Molecular classifications have been developed to characterize the heterogeneity of diffuse large B cell lymphoma (DLBCL), with activation of BCL6 and Notch signaling pathways acting as driving pathogenic features in C1/BN2 subtype tumors. Among C1/BN2 defining variants are truncating/inactivating SPEN mutations. SPEN regulates the Notch signaling pathway by initiating formation of a repressive complex that facilitates transcriptional repression of Notch target genes. Our group has identified SPEN mutations enriched in DLBCL cases failing to achieve event-free status at 24 months (EFS24). This contrasted with generally favorable outcomes in C1/BN2 tumors, suggesting a potential high-risk molecular feature with unexplored significance. Presenting an opportunity to expand underlying mechanisms and risk stratification in C1/BN2 tumors, we aimed to use a functional genomic approach, combined with clinical data, to characterize the alternative biology stemming from SPEN truncating mutations. Methods: FFPE tumor biopsies from newly diagnosed DLBCL cases (N=444) enrolled in the Mayo Clinic/Iowa Lymphoma Molecular Epidemiology Resource (MER) program were analyzed by WES (n=404) and RNA-seq (n=321). Biologic and clinical variables were annotated for all cases. CRISPR/Cas9 introduced truncating mutations to the endogenous SPEN locus in OCI-LY3 & SU-DHL-2 ABC-type human cell lines to establish models of SPEN depletion. Functional in vitro analysis utilized standard methods for RNA-seq, cell proliferation, western blot, and flow cytometry. Results: WES identified 29/404 ndDLBCL (7.2%) harboring SPEN mutations; n=17 missense and n=12 nonsense or frameshift insertions/deletions (truncating variants - [trunc]), the later considered as the focal point of our study. Against all cases, SPEN trunc cases displayed enrichment for high-risk factors including non-GCB COO (p=0.06, OR=3.7, 95% CI [0.9, 22.2]) and primary refractory disease (PTR; p=0.02, OR=5.0, 95% CI [1.05, 19.9]), and demonstrated inferior OS (p=0.06, HR=2.2), where a majority of clinical progression events occurred within 12 months of diagnosis. Leveraging RNA-seq, we observed 11 of 11 SPEN trunc cases exhibiting a double-hit negative gene expression signature, 9/11 cases with inflammatory or depleted microenvironment (LME), and 8/11 with a signature associating with intermediate/high risk of EFS24 failure. Applying the LymphGen algorithm, 5/12 SPEN trunc cases received the BN2 classification, observing co-occurring mutations with CD70 (p&lt;0.01, OR=9.68), BRCA2 (p=0.03, OR=5.84), TET2 (p=0.02, OR=4.91), IRF4 (p=0.02, OR=7.27), and PTPRD (p&lt;0.01, OR=8.31) genes, and were mutually exclusive with ARID1A, BCL2, EZH2, FAS, HLA-B, IRF8, and TMEM30A mutations ( Figure 1A). Isolating SPEN trunc against BN2 cases without SPEN variants (n=14), despite similar clustering of non-GCB COO (p=0.7), PTR was exclusively observed in SPEN trunc cases, accompanied again by inferior OS (p=0.07, HR=4.2; Figure 1B). In the OCI-LY3 SPEN depletion model, GSEA analysis from RNA-seq revealed activation of E2F (NES=2.7; p=9.4E-20) and MYC target genes (NES=2.3; p=4.1E-6), while both OCI-LY3 and SU-DHL-2 models featured depletion of antigen presentation genes (NES= -1.8, p=5.7E-4; NES= -2.2, p=3.8E-7, respectively). A decrease of MHC class II proteins (HLA-DR, HLA-DP) was confirmed by flow cytometry in both models (p&lt;0.01). Hypothesizing loss of SPEN enhances reliance on the BCL6-HDAC3 axis featured in BN2 tumors, we treated both SPEN depletion models with a class-I HDAC inhibitor highly selective for HDAC3, BRD-3308, and indeed observed a 1.4 and 2.4-fold increase in sensitivity compared to WT cells (p=0.07, p&lt;0.01, respectively). Significance: We find SPEN trunc mutations in poor acting DLBCL cases with clinical and biologic characteristics that diverge from C1/BN2 biology. The enhanced rate of clinical progression highlights urgency to better characterize these tumors. Our data suggests loss of SPEN regulatory mechanisms may act cooperatively with C1/BN2 biology to facilitate tumor immune evasion and drive progression through Notch-mediated pathways. Importantly, in vitro evidence shows SPEN deficient lymphoma cells are susceptible to targeted therapeutics against dependent pathways, providing rationale for advancing individualized care strategies for affected patients.