Aggressive B-cell Research Articles

Axicabtagene Ciloleucel in Patients Aged 75 and Older: A Retrospective Cohort Study

Background The treatment of older patients with relapsed/refractory (R/R) aggressive non-Hodgkin B-cell lymphomas (NHL) remains a significant clinical challenge. Chimeric antigen receptor T-cell (CAR-T) therapy represents a potentially curative option, but elderly patients remain underrepresented in relevant clinical trials, and less is known about the safety of CAR-T among patients older than 75 years. Toxicities including cytokine release syndrome (CRS), neurotoxicity (ICANS), arrhythmias, renal failure, and cytopenias may pose higher risk in elderly patients with comorbidities and decreased physiologic reserve compared to younger CAR-T recipients. We sought to characterize outcomes and toxicities of axicabtagene ciloleucel (axi-cel) in a cohort of patients aged ≥ 75 years. Methods This was a retrospective analysis of consecutive patients aged ≥ 75 years with R/R NHL who received axi-cel at New York-Presbyterian/Weill Cornell Medical Center. Data collected included patient characteristics and outcomes including response rates, event-free and overall survival, rates of CRS, ICANS, arrhythmias and severe cytopenias, and length of hospital stay. CRS and ICANS severity were graded following the ASTCT consensus criteria. Cytopenias were graded as per NCI-CTCAE v5.0. Event free survival (EFS) was defined as time to disease progression or death. Results We identified 15 patients with a median age of 78 (range 75-87) years; 6 (40%) were ≥ 80 years of age. 14 patients had a diagnosis of large B-cell lymphoma and 1 had multiply relapsed follicular lymphoma. Ten patients (67%) had received 3 or more prior lines of therapy. Additional baseline characteristics are summarized in Table 1. Among 10 patients (67%) who developed CRS of any grade, only 1 (7%) experienced grade ≥3 CRS; this CRS event was fatal. Five (33%) patients developed ICANS with only 1 patient (7%) developing grade ≥3 ICANS. Two (13%) patients and 10 (67%) patients developed grades 3 and 4 cytopenias within 30 days of infusion, respectively. Four (27%) patients required blood or platelet transfusions within 100 days of infusion. Febrile neutropenia was seen in 8 (53%) patients, and 8 patients had culture or PCR-verified bacterial (33%), viral (40%), or fungal (7%) infections. Only 1 (7%) patient developed a new abnormality on EKG within 30 days of infusion (right ventricular conduction delay). Median length of inpatient stay was 17 days (range 13-35), and 2 (13%) required re-hospitalization within 100 days of infusion. Overall and complete response rates at 100 days post-infusion were 13 (86%) and 11 (73%), respectively. At one year, overall and event-free survival were 72.2% (95% CI: 54.4-99.6%) and 53.3% (95% CI: 33.2%-85.6%), respectively. The median overall survival was 30.8 months with a median follow-up of 21.4 months. One patient experienced treatment-related mortality due to CRS, and 5 (33%) had subsequent disease relapse. Conclusion These results suggest that axi-cel is an effective and well-tolerated treatment for patients age ≥ 75 with R/R NHL. Only 1/15 patients developed grade ≥3 CRS or ICANS which is comparable to reported rates of toxicities in younger patients. Although multiple studies have identified cytopenias and cardiac arrhythmias as major CAR-T toxicities, a minority of our cohort required transfusions, and none developed life-threatening arrhythmias. OS and EFS were also comparable to real-world experience seen in younger patients. This study may be limited by the selection of a relatively fit population of elderly patients; however, the median Karnofsky Performance Score was 80 and ranged as low as 60. Overall, these findings show that axi-cel may be an appropriate strategy for relapsed aggressive NHL in carefully selected patients in their eighth and even ninth decades of life.

A Single Center Experience with Using Dose-Adjusted Repoch in Elderly Patients with Large B-Cell Lymphoma

Background: RCHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) has been the predominant regimen for treatment of aggressive large B-cell lymphomas (LBCL). In the CALGB 50303 trial, REPOCH (rituximab, etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin) was associated with similar efficacy but increased toxicity compared to RCHOP, however pts with higher risk disease by International Prognostic Index (IPI) had longer progression-free survival (PFS) with REPOCH therapy in the subgroup analysis ( J Clin Oncol, 2019). Providers may be hesitant to use REPOCH therapy in older, frail patients (pts) with high-risk LBCL due to concern for increased toxicity. One study demonstrated that empirically dose-reduced REPOCH (30-50% dose reduction by age group) was effective and well-tolerated in an elderly population ( Ann Hematol, 2018).A separate study showed significantly reduced neuropathy rates without compromised efficacy when vincristine doses in the REPOCH regimen were capped at 0.5 mg/d compared to 0.4 mg/m 2/d ( Leukemia & Lymphoma, 2019). At our institution, vincristine doses are capped at 0.4 mg/d for all pts, and REPOCH dosing is not empirically dose-reduced based for age alone. The aim of this study is to provide additional insights on the safety and efficacy of REPOCH in elderly pts diagnosed with LBCL with a routine vincristine dose cap of 0.4 mg/day (“REPOCH 0.4”). Methods: In this single cohort study, pts with newly diagnosed LBCL that received dose-adjusted REPOCH 0.4 at a single institution from August 2015 to October 2020 were retrospectively reviewed. Pts were excluded if they received fewer than 2 cycles, had CNS disease, transferred to another facility during treatment, or were lost-to-follow-up. Primary endpoints included adverse effects (AEs), overall response rates (ORR) via Lugano staging at end of treatment, 2y PFS, and overall survival (OS). Survival endpoints were estimated via Kaplan-Meier survival analysis. Secondary endpoints included CNS relapse rates and percentage of pts electing for hospice during therapy. Outcomes were descriptively compared to the historical cohort (N=241) in the CALGB 50303 trial receiving R-REPOCH without a vincristine dose cap ( J Clin Oncol, 2019) . Results: Forty-six pts were eligible for analysis (“entire cohort”), 19 of whom were 70 years or older (“70+ cohort”, figure 1). Median age in the entire and 70+ cohort was 67 and 75 years respectively. In the entire cohort, 23.9% of pts were double-hit, 43.5% had a non-germinal center phenotype, and 78.3% had stage III-IV disease. The median number of REPOCH 0.4 cycles received in both the entire and 70+ cohort was 6. In the 70+ cohort, 57.9% of pts were escalated to a dose level (DL) of 2 or 3 with no pts escalated beyond DL 3. Fifty-eight percent of pts in the entire cohort received intrathecal CNS prophylaxis and all pts received pegfilgrastim support. Pts in the entire cohort were older and higher-risk in comparison to the historical cohort as evidenced by IPI risk and double-expressor status. In the entire cohort, REPOCH 0.4 was well-tolerated (figure 2) with grade 3+ neuropathy rates of only 2.2% (5.2% in the 70+ cohort). Febrile neutropenia (all grades) occurred in 19.5% and 15.8% of the entire cohort and 70+ cohort respectively. Only 1 patient in the 70+ cohort experienced acute ejection fraction (EF) decline during therapy. ORR (complete and partial response) in the entire and 70+ cohort were 80% and 74% respectively. Complete response (CR) rates in the entire and 70+ cohort were both 63%. 2y PFS in the entire cohort was 68.3% (median not reached), and the 2y OS in the entire cohort was 82.9% (median not reached) which compares favorably to the 2y OS of 86.5% in the historical cohort. Overall, only 7% of pts elected for hospice and only 4% experienced CNS relapse. Conclusion: Pts with LBCL 70+ years tolerated dose-adjusted REPOCH 0.4 mg/d similarly to the entire cohort of all ages indicating that empiric dose reduction for age alone isn't necessary. CR rates were similar (63%) with pts 70+ years compared to pts in the entire cohort of all ages. REPOCH 0.4 mg/d was observed to have numerically lower rates of grade 3 or higher neuropathy (2.2%) compared to vincristine 0.4 mg/m 2/d (8.4%, J Clin Oncol 2019) and vincristine 0.5 mg/d dosing strategies (4.5%, Leukemia & Lymphoma 2019) . To our knowledge, this is the first report of outcomes with REPOCH using a vincristine dose cap of 0.4 mg/d.

A Phase 1, Open-Label, Multicenter, Dose-Escalation Study of Sgr-1505 As Monotherapy in Subjects with Mature B-Cell Malignancies

Background and Significance: MALT1 (Mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of the MALT1-BCL10-CARD11 complex downstream from the Bruton Tyrosine Kinase (BTK) on the B-cell receptor signaling pathway. MALT1 is a key mediator of nuclear factor kappa B (NF-κB) signaling, which is the main driver of a subset of B-cell lymphomas. MALT1 is considered a potential therapeutic target for several subtypes of non-Hodgkin B-cell lymphomas and chronic lymphocytic leukemia (CLL), including tumors with acquired BTK inhibitor (BTKi) resistance. Constitutive activation of NF-κB is a molecular hallmark of activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL), and MALT1 may have utility as a treatment option for ABC-DLBCL. Furthermore, a MALT1 inhibitor (JNJ-67856633) showed efficacy in mature B cell malignancies from phase 1 studies (ref 1, 2). SGR-1505 is an oral potent small molecule allosteric inhibitor of MALT1 that inhibits MALT1 enzymatic activity and demonstrates anti-proliferative activity in BTKi-sensitive (OCI-LY10) and BTKi-resistant (OCI-LY3) ABC-DLBCL cell lines. SGR-1505 administered as a single agent and in combination with the approved Bruton's tyrosine kinase (BTK) inhibitor, ibrutinib, demonstrates tumorostatic and regressive antitumor activity in ABC-DLBCL cell line-derived and patient-derived xenograft models. These data suggest that SGR-1505-mediated MALT1 inhibition may expand therapeutic options for patients with selected B-cell lymphomas, supporting further evaluation of SGR-1505 in clinical trials. Study Design and Methods: SGR-1505-101 (NCT05544019) is a phase 1, multicenter trial of SGR-1505 as monotherapy in subjects with mature B-cell malignancies (figure 1). At present, the study is open to accrual at multiple investigative sites in the US with plans to expand to Europe. The primary objective is to evaluate safety and tolerability of SGR-1505 as monotherapy and to identify the maximum tolerated dose (MTD) and/or recommended dose (RD). Secondary objectives are to evaluate the pharmacokinetic (PK) profile, food effect, drug-drug interaction, and preliminary anti-tumor activity of SGR-1505. Key inclusion criteria are: history of mature B-cell malignancy (including aggressive and indolent B-cell lymphomas, Waldenström macroglobulinemia, and CLL); measurable or detectable disease according to the applicable disease-specific classification system (Lugano, iwCLL, WWM6); Eastern Cooperative Oncology Group (ECOG) performance status of ≤ 2. Patients with indolent B-cell lymphomas or CLL must have an indication for treatment and not require immediate cytoreductive therapy. Subjects with symptomatic or active CNS involvement, and other conditions or laboratory findings placing them at increased risk to the use of an investigational drug are excluded. SGR-1505 is initially dose-escalated using an accelerated titration design in cohorts of 1-6 subjects, and at higher dose levels using a conventional 3+3 design.

Multi-Cancer Early Detection Test Is Sensitive and Accurate in Detecting Shared DNA Methylation Signal in a Variety of Lymphoid and Plasma Cell Neoplasms

Background: Recent technological advances in molecular diagnostics have facilitated development of blood-based multi-cancer early detection (MCED) tests intended to complement standard of care single-cancer screening. Implementing MCED at population scale has the potential to improve survival for patients with lymphoid neoplasms (LyN) and plasma cell neoplasms (PCN). The Galleri test, launched in 2021, is the first commercially available liquid biopsy MCED test in the United States that has been validated.Galleri is a genome-wide targeted methylation sequencing test that detects cancer-related DNA methylation changes in cfDNA that are shared by many cancers. By targeting 100K fragments in the genome and ~1 million CpGs, processed by a machine learning classification algorithm, Galleri is able to not only detect the presence of a cancer signal, but also to identify the most likely origin of the cancer signal (CSO) in a tissue or organ. Here we report specific performance of Galleri in LyN and PCN, reported as either lymphoid or plasma cell CSO from two large clinical studies and real-world implementation (RWE) among 100,000 individuals. Methods: The case-control (CCGA3) Circulating Cell-free Genome Atlas study (NCT02889978) included 2,823 cancer participants (cases, pre-treatment) and 1,254 non-cancer participants (controls), the prospective interventional PATHFINDER study (NCT04241796), included 6,578 asymptomatic individuals aged ≥50 and received an earlier version of MCED to assess the feasibility of implementing MCED testing. With more than 100,000 results returned to providers across the US to date, we also report early data from systematic collection of outcomes for cases with a Cancer Signal Detected (CSD) result via a controlled quality assurance (QA) program to monitor MCED testing in a real-world population. Results: In CCGA3, the overall sensitivity of Galleri in LyN was 52.9% (119/225; 95% CI 46.4%, 59.3%); the overall sensitivity in PCN was 72.3% (34/47; 95% CI 58.2%, 83.1%) ( Table 1). Galleri can detect a shared cancer signal from a variety of lymphoid and plasmacytic neoplasms, including Hodgkin lymphomas (HL) and non-Hodgkin lymphomas; B- and T-cell neoplasms; from mostly indolent follicular lymphoma (FL) to aggressive Diffuse Large B-Cell Lymphomas (DLBCL) and Multiple Myeloma ( Table 1). Sensitivity was higher in more aggressive lymphomas, like DLBCL (70.5%). Galleri demonstrated excellent performance in hematologic cancers predicting lymphoid CSO with 99% accuracy and plasma cell CSO with 100% accuracy in true cancer cases. Galleri is intended to detect bona fide cancers and not preneoplastic conditions (ex. MGUS, MBL, CHIP). The sensitivity increased from stage I to stage IV, likely reflecting higher tumor burden and tumor fraction in cfDNA ( Table 2). The sensitivity for early combined stage I and II cancers, which are more responsive to treatments, was 45.7%. In PATHFINDER, using an updated commercial version of the test, there were 11 cases with CSD result and a top CSO prediction consistent with a hematologic malignancy: 7 lymphoid and 4 plasma cell neoplasia. A total of 7/11 (63.6%) participants had hematologic malignancies diagnosed: 6 lymphomas (stages I-IV) and 1 PCN. Galleri demonstrated a high PPV of 54.5% for cases with predicted CSO either LyN or PCN, and 71.4% for cases with predicted CSO Lymphoma. Early clinical outcomes data from a controlled QA quality assurance (QA) program collected from an initial limited subset of CSD cases with follow up information showed that, similar to our clinical trial data, this test is able to detect a shared cancer signal in a variety of lymphomas (chronic lymphocytic leukemia [CLL], DLBCL, FL, PTCL, HL), some in early stages, such as HL stage I. More detail on the MCED real-world experience will be presented at the meeting. Conclusions: Findings from two large validation studies and RWE demonstrate that Galleri promises to be a state of the art molecular tool that detects a shared cancer signal and is able to identify a large variety of histopathologic entities of lymphoid and plasmacytic origin, which currently have no other standard of care screening regimen. This tool may lead to better treatment outcomes and decreased mortality saving lives through the early detection of these previously unscreened cancers.

Efficacy and Safety of Chimeric Antigen Receptor T Cells Therapy Combined with Zanubrutinib in the Treatment of Relapsed/Refractory Diffuse Large B-Cell Lymphoma

Background: While complete response rate of CD19 CAR-T in diffuse large B-cell lymphoma (DLBCL) reached 40-60%, about 30%-50% of patients still face relapse, strategies that can enhance the therapeutic effects of CAR-T cells infusion should be actively explored. Bruton's tyrosine kinase (BTK) is an essential component of multiple signaling pathways including B-cell receptor signaling that regulate B cell proliferation, survival, and functions. These receptor signaling has been implicated in the survival of malignant B-cells making it a promising therapeutic target for various B-cell malignancies and inflammatory diseases. Different types of BTKi regulate T cell and B cell function through different signal pathways. Research has proved that CD19 CAR-T Cells in combination with ibrutinib for the treatment of relapsed/refractory aggressive B-cell lymphomas shows a high response rate and low level of adverse events, while zanubrutinib has not been reported. Therefore, our center explored the efficacy and safety of CD19 CAR-T Cells in combination with zanubrutinib for the strategy of relapsed/refractory DLBCL. Patients and Methods: The study included 6 r/r DLBCL patients, 4 patients have previously received 3-line or above treatment, and 2 patients began CAR-T treatment after 1-line chemotherapy. All patients received fludarabine (FLU) and cyclophosphamide (CTX) conditioning chemotherapy (FLU 30mg/m 2. d1-3; CTX 500mg/m 2, d1-3) before CAR-T infusions. T cells were apheresis collected and transduced with a lentiviral CD19 scFv CAR fused with intracellular signaling domains: CD28/CD27/CD3z-iCasp9. CAR-T cells were infused at dose of 2.0x10 6 cells/kg. The quality of apheresis cells, gene transfer and T cell proliferation efficiencies, and effective CAR-T infusion dose were quantitatively scored and documented. All patients began to combine with zanubrutinib after one month of CAR-T cells infusion. Results: After one month of CAR-T cells infusion, total of 50% (3/6) of patients achieved complete response (CR), while 50%(3/6) of patients were evaluated as partial response (PR), with an overall response rate (ORR) of 100%. Within 6 months after combined with zanubrutinib, all patients of PR achieved CR. In the evaluation of safety, 83% (5/6) of patients occurred grade 2 cytokine release syndrome (CRS), and 33.3% (2/6) patient occurred grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS), no case of severe CRS defined as≥grade 3 occurred, 66.7% (4/6) of patients occurred grade 2 hematotoxicity and 33.3% (2/6) of patients occurred grade 4 hematotoxicity. After a median follow-up of 19.5 months, 6 patients maintained CR, with an duration of response (DOR) of 100%, median DFS and OS were not reached. All patients can be detected CAR-T cells sustain in peripheral blood mononuclear cells continuously until last follow-up date(Fig.2). Continued follow-up will confirm whether the CAR-T cells therapy combined with zanubrutinib can obtain long term overall survival in these study. Conclusions: Our study proved that CD19 CAR-T Cells combined with zanubrutinib for the strategy of relapsed/refractory DLBCL and as maintenance treatment showed strong efficacy with acceptable toxicity, the current results already support that zanubrutinib in the treatment of r/r DLBCL is expected to reduce the risk of relapsed, thereby prolonging the disease-free survival of patients, without increasing the incidence of adverse effects. More follow-up and clinical trials are needed to clarify the efficacy of Chimeric Antigen Receptor T Cells Therapy combined with zanubrutinib in the treatment of r/r DLBCL.

Hnrnpu mutations Are Haploinsufficient and Alter the Transcriptome of MYC-Driven Lymphomas

Introduction: Burkitt lymphoma (BL) is an aggressive B-cell cancer, characterized by translocations that juxtapose a potent immunoglobulin enhancer with the MYC oncogene. MYC translocations are also found in 10% of tumors with diffuse large B-cell lymphoma morphology. When a BCL2 translocation is also present, these high-grade B-cell lymphomas (HGBCL-DH- BCL2) have poor prognosis. While sustained MYC expression can promote cell activation and proliferation, it also has potent and acute effects on programmed cell death, which cancer cells must overcome in order to survive. Identification and functional characterization of common mutations that cooperate with MYC are thus important for understanding its role in pathobiology. Methods: Patient samples were obtained from an ongoing meta-analysis of mature B-cell neoplasms including unpublished data from the Lymphoma/Leukemia Molecular Profiling Project. Simple somatic mutations were identified from whole genome or exome sequencing data using an ensemble of variant callers. MYC rearrangements breakpoints were identified using GRIDSS and Manta. All functional studies were performed in an EBV+ BL cell line (Raji). HNRNPU mutations were introduced into cells using IDTs ALT-R CRISPR-Cas9 system, clonally isolated, and verified by sequencing. Subsequent RNA-sequencing was performed. Knockdown and overexpression experiments were performed using an HNRNPU expression vector or siRNA targeting endogenous HNRNPU. HNRNPU eCLIP in HepG2 and K562 cells were obtained from the ENCODE project consortium. Differential gene expression was analysed with DESeq2. Peak-calling and individual crosslink sites were detected using PureCLIP. Results: Within the sequencing data, we noted that several RNA binding proteins were commonly mutated in BL and HGBCL-DH- BCL2. Mutations affecting HNRNPU were identified in tumors from 11.6% of HGBL-DH- BCL2 and 5.6% of BL patients. These mutations were predicted to be inactivating but, unlike conventional tumor suppressor genes, an inactivating mutation on the other allele was never observed. HNRNPU mutations were significantly enriched in EBV+ BLs. The gene product (hnRNP U) is an RNA- and DNA-binding protein that plays a central role in gene expression regulation. To understand their biological effect in aggressive lymphomas, we introduced heterozygous inactivating mutations in the most affected region of HNRNPU. Clones with confirmed knockout exhibited reduced expression at the mRNA and protein level. Through gene expression analysis, comparing the parental to two mutant lines, we identified 615 differentially expressed genes (358 downregulated, 219 upregulated). Pathway enrichment analysis revealed reduced expression of genes in several relevant pathways such as MYC, TP53 and DNA damage response. This was consistent with our observation of reduced MYC protein in the mutant lines. A role of hnRNPU-in MYC modulation was further in experiments when HNRNPU knockdown reduced and overexpression increased MYC expression. Overexpression of hnRNP U also resulted in cellular stress and a decrease in cell proliferation. Using actinomycin D chase experiments, we found that hnRNPU loss leads to reduced MYC transcript stability. Crosslinking immunoprecipitation suggests that hnRNP U binds directly to the MYC transcript in poly G tracts in intron 1. This region is predicted to fold into G-quadruplexes, a secondary structure that can be bound by hnRNP U. HNRNPU mutations are only observed in tumors with an intact MYC locus rather than those with a rearrangement in intron 1. Taken together, these findings suggest that the region of MYC upstream of intron 1 may be relevant for HNRNPU-mediated modulation of MYC. Conclusion: HNRNPU mutations are novel recurrent driver mutations specifically within MYC translocated B-cell lymphomas. HNRNPU acts as a modulator of MYC expression, and the most common mutations are predicted to negatively impact this role. We propose a model where HNRNPU mutations moderate MYC expression, thus buffering MYC-induced apoptosis and proteotoxic stress. Further experiments to explore the role of HNRNPU binding on MYC expression and the potential role in buffering the proteotoxic stress associated with MYC translocations are ongoing. Insights into the mechanisms contributing to disease will support ongoing work to establish in vitro and in vivo models for future therapeutic investigations.

A Compilation of Experiences in Utilizing CAR-T Cell Therapy for Richter's Transformation

Objective:Even with Bruton tyrosine kinase inhibitors (BTKis) and BCL2 inhibitors, the prognosis for Richter's Transformation (RT) remains dismal, with a median overall survival (OS) of only 9-12 months from initial diagnosis. Although there are reports on CAR-T therapy for RT, the sample size is small and the conclusions vary greatly. Methods: We conducted a retrospective investigation of the efficacy of CAR-T cell therapy in treating RT at our center. Each participant provided their informed consent to partake in the “Phase II Clinical Trial of Sequential CAR-T Cell Therapy Targeting Different B-Cell Epitopes for Recurrent, Refractory Aggressive B-Cell Lymphoma”(Protocol number: BR-XG-CART-II, ChiCTR2100055062, Ethical Approval Number 20191225-PJ-003). Before lymphocyte collection, chemotherapy regimen without fludarabine/ Bendamustine is first used to reduce peripheral blood tumor load if peripheral blood tumor cells are higher than 10%. Patients with extramedullary tumor loads of 5cm or larger were treated with tumor reduction chemotherapy between lymphocyte collection and CART cell transfusion. The pretreatment scheme based on Bendamustine or FC scheme was adopted before CAR-T cells were infused. The efficacy and safety of CART cells were observed. Results: Between Feb 1, 2020 and Feb 28, 2023, our center administered CAR-T cell therapy to 16 patients diagnosed with RT, all of whom developed into diffuse large B-cell lymphoma. The patient population had a median age of 59 years (range: 42-69 years), consisting of 62.5% (10/16) males and 37.5% (6/16) females. The ECOG scores varied between 0 and 3. The median number of prior treatment lines was 5 (2-7). 93.8% failed previous BTKis treatment and 75% failed BCL2 inhibitor treatment; 93.8% had undergone chemotherapy (median 6 courses). 1 patient had previously failed universal CAR-T cell therapy, and another relapsed after autologous transplantation. 90% had at least one TP53 abnormality detected by either FISH or second-generation gene sequencing. Extramedullary masses were found in 87.5% (14/16) of patients, with a median maximum diameter of 42.5mm (18-144mm). Masses larger than 5cm were found in 25% (4/16) of cases, marrow involvement was noted in 62.5% (10/16), and cerebrospinal fluid was affected in 18.75% (3/16). None of the patients had central masses observed in their MRIs. During lymphocyte collection, 58.3% of patients showed positivity for peripheral blood tumor cells, with a maximum tumor cell proportion of 5.69%. The median infused CAR-T cell dose was 1.37×10 6/kg. 12 of them were injected with mouse CD19CAR-T, 2 with human CD20CART, and 2 with human CD19CART. CRS occurred in 81.3%of patients, of which, 68.8%were grade 1-2 and 12.5%were grade 3. Central CRS occurred in 6.3% (1/16) of patients. The complete response (CR) rate at the 3-month mark stood at 56.3% (9/16), with an overall response rate (ORR) of 68.8% (11/16). As of July 6, 2023, the median follow-up time was 10.6 months (4.4-33.6m), the 1-year OS was 73.4%, and the 1-year PFS was 68.8%. Median OS and PFS were not reached. One of the five patients who did not respond later underwent allogeneic hematopoietic stem cell transplantation and is still alive. The other four, despite attempts at salvage treatment including second CAR-T cell therapy, all died, with a median OS of only 5.6 months (4.4-8.9m). None of the four patients with extramedullary masses larger than 5cm achieved ORR despite tumor-reducing chemotherapy prior to CAR-T infusion. Only 1 of 3 patients with central involvement achieved CR and experienced grade 3 CRS along the way, while the other 2 did not respond to CAR-T therapy. Among all patients, only one experienced central CRS, and his cerebrospinal fluid showed no signs of tumor cells prior to the administration of CAR-T treatment. A patient who was in CR succumbed to an acute coronary event 19 months following infusion, despite remaining in CR. Conclusion: Even in the presence of TP53 gene abnormalities, resistance to BTK/BCL2 inhibitors, and unsuccessful previous chemotherapy regiments, A single CAR T cell therapy can result in CR in more than half of RT patients. Nonetheless, the prognosis remains bleak for patients presenting with large tumor masses and central nervous system involvement, despite having received chemotherapy aimed at reducing tumor size. The outlook is particularly dismal for those patients who fail to respond to CAR-T cell therapy.

Thiotepa, Busulfan and Fludarabine Conditioning Regimen in Adult Patients Undergoing Allogeneic Stem Cell Transplantation for Lymphoid Malignancies. a Study on Behalf of Geth-TC

Background: The optimal conditioning regimen in allogeneic hematopoietic stem cell transplant (alloHCT) for lymphoid malignancies remains unclear. Reduced intensity regimens (RIC) allow older and more comorbid patients to receive alloHCT since lower non-relapse mortality (NRM) is observed, but disease control may still be a concern. The use of reduced toxicity conditioning regimens allows better disease control with a cost of higher toxicity. To date, no data are available regarding the use of thiotepa, busulfan and fludarabine (TBF) in adult patients allografted for lymphoma. Our aim is to describe survival outcomes and treatment-related toxicities of patients with lymphoid malignancies conditioned with TBF. Methods: Between December 2011 and July 2022, 116 patients diagnosed with lymphoid malignancies received a first alloHCT conditioned with a TBF-based regimen in 9 Spanish institutions. All consecutive patients were included in the study and data were collected retrospectively. Low-dose TBF was defined as the use of 5 mg/kg of thiotepa (T5) while a high-dose TBF was considered as the administration of 10 mg/Kg of thiotepa (T10). Main outcome variables were overall survival (OS), progression-free survival (PFS), NRM, relapse incidence (RI), cumulative incidence of acute (aGVHD) and chronic GVHD (cGVHD), and hematological recovery. Probabilities of OS and PFS were calculated using the Kaplan-Meier estimator method, and NRM, RI, GVHD and hematological recovery as cumulative incidences. Results: Baseline characteristics are summarized in Table 1. The median age was 52 (range 23-70) years and 62.1% of the patients were males. Aggressive B-cell lymphoma was the most common diagnosis (43.1%). Seventy-seven (66.4%) patients received T10, and 39 (33.6) T5. Median follow-up among survivors was 46.9 (range 16.9-136.5) months. At 3 years, OS, PFS, NRM and RI were 54.7% (95% confidence interval [CI], 45.5-63.9%) ( Figure 1), 44.9% (95% CI, 35.7-54.1%), 34.9% (95% CI, 27.1-44.9%) and 20.2% (95% CI, 14-29.2%), respectively. Of the 53 patients who died during follow up, causes of death were: 12 relapses and 41 transplant toxicities (16=infections, 8=GVHD, 5=endothelial complications, 1=traumatism, 11=missing). Fifty-one percent of toxicity-related deaths were observed during the first 3 months from transplant. All but 2 patients engrafted. The cumulative incidence of neutrophil engraftment at day +30 was 91.4% (95% CI, 86.3-96.8%). The cumulative incidence of grade II-IV aGVHD at day +100 was 25.4% (95% IC, 18.5-34.9%), with 8 patients presenting grade III-IV aGVHD. The 3-years cumulative incidence of all grade cGVHD was 29.8% (95% CI, 22.5-39.6%), with 15 patients presenting with moderate-severe cGVHD. Seventeen patients developed venooclusive disease (14.7%) with a median time from alloHCT infusion of 14 (range 7-93) days. On univariate analysis, no prognostic factors were found in terms of NRM, OS and PFS. However, higher doses of thiotepa were associated to a lower relapse incidence (13.2% vs 35%, p=0.0075) without differences in terms of NRM. The use of myeloablative conditioning (vs RIC) showed a trend towards a lower relapse incidence although it was not statistically significant (9.4% vs 24.5%, p=0.059) with no impact in NRM rates. On multivariate analysis these results could not be confirmed. Conclusions: In our series, TBF conditioning regimen allows efficient disease control at the expense of increased incidence of severe early toxicities. Despite higher NRM, TBF conditioning regimen showed similar OS rated compared to other regimens used for lymphoid malignancies. This regimen could be considered for fit patients with high-risk lymphoid diseases.

PD-1 Blockade Combined with R-CHOP in Patients with Newly Diagnosed EBV-Positive Diffuse Large B-Cell Lymphoma: A Retrospective Analysis

Background Epstein-Barr virus (EBV) -positive diffuse large B cell lymphoma (EBV+ DLBCL) is a relatively rare aggressive B-cell lymphoma. Most patients present with advanced disease, high incidence of extranodal involvement of 30% to 80% (Keane, Eur J Haematol, 2019), as well as poor prognosis when treated with immunochemotherapy. However, no consensus on standard treatment has been established by now. It was found that the expression of programmed death ligand-1 (PD-L1) was increased in EBV+ DLBCL (Kim, Cancer Res Treat, 2019). In addition, chronic EBV infection results in T-cell exhaustion that can be reversed by PD-1 blockade. Compared with PD-L1-positive EBV-negative DLBCL cell lines, the application of PD-1 blockade in PD-L1-positive EBV+ DLBCL cell lines can significantly restore T cell proliferation and activity(Quan, PLoS One, 2015), suggesting that targeting the PD-L1/PD-1 pathway represents a potential therapy for EBV+ DLBCL. Therefore, we retrospectively analyzed the data of nineteen patients with EBV+ DLBCL who were treated with PD-1 blockade combined with RCHOP in our center, hoping to provide reference for subsequent research. Methods Eligible pts had treatment-naive histologically confirmed diagnosis of EBV+ DLBCL. Additional eligibility criteria included at least one measurable lesion, acceptable haptic and hematological function. Pts with primary CNS lymphoma, primary mediastinal large B-cell lymphoma, secondary CNS involvement were excluded. Additional exclusion criteria included history of immune system disease, uncontrolled infection. Once deemed eligible, pts will receive six cycles of RCHOP combined with PD-1 blockade(21 days per cycle) as induction therapy and subsequently maintenance with PD-1 blockade for 1 year(21 days per cycle). End of treatment(EOT) responses were assessed by PET-CT according to the Lugano Classification 2014. The primary objective was to assess the overall response rate(ORR) at EOT. Results Between May 1, 2019, and Sep 1, 2022, 19 pts were enrolled. Pts' baseline demographics, clinical and pathological characteristics are shown in Table 1. The median age was 64 years old without significant gender difference. The majority of pts were intermediate- to high-risk by IPI. EBV DNA can be detected in blood serum at diagnosis in more than half of all patients. No patient was diagnosed with double expressor lymphoma among 18 specimens that were detected c-Myc and BCL-2 with immumohistochemical staining. EBV-encoded RNA (EBER) was positive(>80% of the tumor cells) in all specimens. PD-L1 positivity(>10% of the tumor cells) or CD30 positivity(>5% of the tumor cells) was also quite common. Besides, no double hit lymphoma was confirmed in patient with Fluorescence situ hybridization test(n=13). Treatment course and efficacy evaluation of all pts are summarized in Figure 1. All patients enrolled in this study had completed 6 cycles of induction therapy. Totally, the ORR at EOT was 94.7%, and all achieved CMR. The ORR of patients with PD-L1 positivity(n=10) was numerically higher than that of patients with negative PD-L1 expression (n=5) (100% vs 80%). EBV DNA detected in blood serum of thirteen patients have decreased to undetectable level after induction therapy. The immune-related adverse events (irAEs) were emphatically analyzed. One patient had recurrent fever after every dose of PD-1 blockade without obvious evidence of infection. Another patient developed grade 3 diarrhea since the second cycle of PD-1 blockade maintenance. Colonoscopy showed mucosal swelling and multiple superficial ulcers in the whole colon, which indicated immune colonitis. Maintenance therapy was therefore discontinued. No irAE was observed in the rest of patients. With a median follow-up time of 13.1m (7.2-38.5m), the median PFS and OS were not reached. Both of 1 year OS and PFS was 89.5%. Overall, 2 pts experienced disease progression (1 at EOT, 1 at 2 months after ASCT), 2 pts died (1 due to progressive disease, 1 due to COVID-19 infection ). Conclusions PD-1 blockade combined with R-CHOP as induction therapy in patients with newly diagnosed EBV+ DLBCL has demonstrated satisfactory response and limited irAEs. Longer follow-up is still warranted to confirm the long-term benefit, especially for patients with maintenance of PD-1 blockade. Still, we believe this therapeutic strategy can be regarded as a successful attempt at improving prognosis for pts with EBV+ DLBCL.

Inferred Gene Expression By Cell-Free DNA Profiling Allows Noninvasive Lymphoma Classification

Background : Current diagnostic criteria for mature B-cell tumors require a combination of histopathological, immunophenotypic, cytogenetic, and genetic evaluations on tissue biopsy specimens (Campo, Blood 2022; Alaggio, Leuk 2022). Despite this battery of techniques, substantial heterogeneity remains in specific subgroups that can be further resolved using additional molecular diagnostic methods, including gene expression profiling (GEP). For example, among aggressive B-cell tumors, a subset recognized as high-grade B-cell lymphomas can be identified as harboring MYC+BCL2 lesions by FISH (HGBCL-DH-BCL2). However, a significant remaining high-risk subset also have a characteristic tumor gene expression “double-hit signature” or “dark zone signature” (DZsig) (Ennishi, JCO 2018; Alduaij, Blood 2023). Yet, inadequate tissue specimens can limit the application of these techniques for some patients. Separately, the invasive nature of tissue biopsies and their attendant risks can preclude adequate diagnostic evaluations in some patients, and logistical barriers for obtaining surgical tissue specimens can result in diagnostic delays. To address these unmet needs, we tested the performance of a novel noninvasive strategy to detect and classify mature B-cell tumors using plasma cfDNA, with special attention to HGBCL-DH-BCL2 and DZsig. Methods : We used EPIC-Seq to infer expression levels for genes of interest, using cfDNA fragmentomic signals at their transcription start sites (Esfahani, Nat Biotech 2022). We first curated and catalogued genes from prior GEP studies (n=56) of >10k diverse lymphoid tumors profiled by various RNA techniques. We systematically prioritized genes recurrently identified in multiple studies of tumor-specific expression profiles as compared with other tumor types and normal tissues. We included canonical markers used for immunodiagnosis by IHC and FACS, as well as key lineage and differentiation markers of normal B and T cells, and other leukocyte subsets. Finally, we also included recurrently mutated genomic regions and fusion hotspots for quantifying ctDNA levels using variant allelic fractions (VAF). To validate performance of DZsig in FFPE for HGBCL-DH-BCL2, we analyzed 95 FFPE biopsies by FISH (20% HGBCL-DH-BCL2), IHC, and RNA-seq. We then analyzed 36 corresponding pretreatment cfDNA samples (28% HGBCL-DH-BCL2) and used EPIC-Seq to infer gene expression. Results: Starting from our curated catalogue, we designed a targeted lymphoid EPIC-Seq panel (1676 genes; 2.6 MB) to resolve 11 major mature lymphoid malignancies and their clinically relevant molecular subtypes (cHL, DLBCL, HGBCL, FL, MCL, BL, MZL, PMBCL, SLL/CLL, PTCL, and MM). We next applied this EPIC-Seq panel to profile cfDNA samples from 207 lymphoma patients, including cHL (n=113), DLBCL (n=66), FL (n=15), and MCL (n=13) as well as healthy adults to assess specificity. We tested histology specific signatures derived from several existing RNA GEP datasets for cHL from sorted HRS cells (~85k by scRNA-Seq) and for DLBCL (n=959), FL (n=635), and MCL (n=100) from bulk RNA-Seq. Using this approach, we found high correlations between EPIC-Seq signature scores from cfDNA and corresponding circulating tumor VAFs in each histology [cHL (R p=0.83), DLBCL (R p=0.83), FL (R p=0.87), MCL (R p=0.81); Fig A]. In addition, when measuring these tumor derived signatures in cfDNA, each histology was significantly distinguishable from healthy controls (AUCs: cHL 0.94, DLBCL 0.91, FL 0.77, MCL 0.96). We next evaluated the DZsig when measured in LBCL patients and comparing paired tumor and cfDNA samples. We first confirmed the performance of the DZsig for detection of HGBCL-DH-BCL2 in 95 FFPE tumors by RNA-Seq (p<0.0001, AUC 0.85). We then noninvasively measured the DZsig in plasma cfDNA by EPIC-Seq (cfDZsig) and found significant discrimination of HGBCL-DH-BCL2 and DLBCL (p=0.0088, AUC 0.78, Fig B). Conclusions: These results confirm that inferred gene expression by cfDNA profiling allows noninvasive diagnosis of multiple lymphoma subtypes, including classification of challenging diagnostic entities such as HGBCL and DZsig+ tumors. Our ability to design and validate a pan-lymphoid gene panel for detection and classification of several lymphoma subtypes suggests the promise of this approach for other diverse tumors as well as the refinement of cfDZsig using a larger cohort.

Methotrexate in Combination with Orelabrutinib, Thiotepa, and Rituximab (R-MTO) Followed By Autologous Stem-Cell Transplant for Primary Central Nervous System Lymphoma Treatment

Background: Primary central nervous system lymphoma (PCNSL) is a rare, aggressive B-cell malignancy. Despite the possibility of a cure, the prognosis remains poor. The standard treatment includes high-dose methotrexate and rituximab, followed by consolidation with either whole-brain radiation therapy or autologous stem cell transplantation (ASCT). However, there is still no clear consensus on the best treatment approach. To improve outcomes, we incorporated thiotepa chemotherapy, which has shown promising activity in inhibiting tumor growth by disrupting DNA bonds, into the regimen. Additionally, we introduced orelabrutinib, a novel and potent BTK inhibitor with high cerebrospinal fluid concentrations, which has demonstrated efficacy in treating PCNSL. In light of these findings, we conducted a clinical study to evaluate the efficacy and safety of a combination regimen of thiotepa, orelabrutinib, and methotrexate with rituximab (R-MTO) followed by autologous hematopoietic stem cell transplantation (HSCT) in patients with PCNSL. Methods: This is an ongoing, single-center, single-arm, open-label study conducted between April 2022 and July 2023. The study enrolled patients aged 18-80 years diagnosed with PCNSL. The patients received 5-8 cycles of R-MTO regimen as induction therapy, consisting of rituximab 375 mg/m 2 iv on day 0, MTX 3.5 g/m 2 iv on day1, thiotepa, 30-40 mg/m 2 on day 4 and orelabrutinib 150 mg qd po for 3 weeks per cycle). After completing the R-MTO therapy, patients were proposed to receive orelabrutinib monotherapy as maintenance treatment for up to 2 years. The efficacy was assessed by MRI/PET per the International PCNSL Collaborative Group (IPCG) criteria. Patients who did not experience disease progression subsequently underwent consolidation treatment with high-dose chemotherapy (HCT) plus ASCT. The HCT regimen involved busulfan 3.2 mg/kg on days -5, -4, thiotepa 250 mg/m 2 on days -7 and -6, cytarabine 2g/m 2 q12h on days -3 and -2, followed by reinfusion of stem cells on day 0. The primary endpoint of the study was progression-free survival (PFS) at 2 years, evaluated by intention-to-treat (ITT). Secondary endpoints included complete response (CR) rate, overall response rate (ORR), overall survival (OS) and toxicities. Results: Between April 2022 and July 2023, a total of 19 patients with PCNSL were enrolled in our study, including 16 who were newly diagnosed and had not received any prior treatment. The median age of the patients was 62 years, ranging from 31 to 72, with 9 out of 19 (47.37%) being male. By the data cutoff, all the patients had completed 4 cycles of treatment. Additionally, 8 out of 19 (42.11%) patients had completed six cycles of treatment, and 2 out of 19 (10.52%) patients had completed 7 to 8 cycles of treatment. The ORR was 94.73% (18/19), and the CR rate was 89.47% (17/19) after 4 cycles of treatment. However, one patient with a P53 mutation progressed after 2 cycles of treatment and unfortunately died 3.8 months after diagnosis. Among the six patients who underwent autologous HSCT, all of them (100%) remained in CR. One patient died due to a COVID-19 infection after 6 cycles of R-MTO and ASCT. The most common hematological adverse events associated with the R-MTO regimen were mild to moderate (grade 1-2) decreases in neutrophil count, which typically resolved within a few days. Conclusions: The R-MTO induction treatment has demonstrated notable efficacy in achieving higher response rates among patients with PCNSL, and the associated adverse events are manageable.

Infections in Lymphoma Patients Treated with Bispecific Therapies: A Systematic Review and Meta-Analysis

CONCLUSION BACKGROUND Bispecific antibodies (BsAb) demonstrate efficacy in newly diagnosed and relapsed/refractory lymphomas. BsAb act by forming immune synapses between an effector cell (T cell or NK cell) surface markers and target cell surface markers, typically CD19 and CD20 in B-cell lymphoma; and CD30 in T-cell lymphomas. Improved understanding of the infection risks associated with BsAbs is required to improve patient outcome. METHODS MEDLINE, EMBASE, Cochrane and Google Scholar were searched to identify relevant studies up to June 15, 2023. Phase I-III clinical trials and observational studies of BsAb monotherapy and combination therapy in adult patients were included. Study identification, data-extraction and bias-assessment were performed by two authors (Figure 1). The primary outcome was the number of bispecific-treated lymphoma patients who experienced ≥ 1 infection of any grade. Secondary outcomes included severe infections (grade ≥ 3), fatal infections and the incidence of microbiologically confirmed bacterial, viral and fungal infections. Sub-analysis by malignant target (CD19 vs CD20), and monotherapy versus combination therapy was performed. Meta-analysis of proportions estimated the pooled infection incidence using random effects model (Mantel-Haenszel). Cochran's Q test examined heterogeneity. PROSPERO registration (CRD42023433207). RESULTS Of 1796 studies, 23 studies (1423 pts) were included, with a median age of 64 years (IQR 60 - 66). Nineteen studies (1124 pts) reported the primary outcome of all-grade infections, which occurred in 37% of patients (95%CI: 30 - 45%) (Figure 2). 13% of patients experienced a grade ≥ 3 infection (12 studies, 843 pts, 95%CI: 9 - 17) and 2% (N = 35) experienced a fatal infection (18 studies, 1318 pts, 95%CI: 1 - 2%). 35 fatal infections were reported (1124 pts), with aetiology reported in 97% (N = 34). 68% of fatal infections (23/34) were microbiologically identified. Fatal infections were predominately viral (70%, 16 infections, 15 deaths from COVID-19, 1 death from EBV), followed by fungal (17%, 4 infections, 3 deaths from pneumocystis jirovecii, 1 death from candida species), bacterial (9%, 2 infections, E. coli and Klebsiella sepsis) and 1 parasitic infection (toxoplasmosis). 11 clinically defined fatal infections occurred; 3 fatal pneumonias, and 8 cases of fatal ‘septic shock’ without a causative pathogen listed. 302 patients (8 studies) with aggressive B-cell lymphoma were compared to 129 indolent lymphoma patients (2 studies). All-grade infections were significantly higher in the aggressive B-cell lymphoma subgroup (41%, 95%CI: 33 - 49%) compared to the indolent subgroup (27%, 95%CI: 10 - 44%, z = 2.77, p = 0.005). The weighted median of prior therapies in the aggressive lymphoma group was comparable to the indolent group (3 vs. 2.5 prior therapies). Grade ≥ 3 infections (14% vs 11%) and fatal infections (3% vs 0%) did not differ significantly between aggressive and indolent subtypes (p > 0.05). Patients receiving CD20/CD3 BsAb monotherapy experienced a significantly higher rate of all-grade infections (42% vs 29%, z = -3.14, p < 0.01) and grade ≥ 3 infections (17% vs 7%, z = -3.23, p < 0.01) than patients receiving CD19/CD3 monotherapy. Fatal infections were comparable between CD20/3 and CD19/3 products. Microbiological aetiology and clinical sites of infections were not commonly reported. In subanalysis of CD20/CD3 products, pooled all-grade infections did not differ significantly between monotherapy epcoritimab (34% 95%CI: 21 - 47%) and glofitamab (39% 95%CI: 21 - 56%). Analysis of all-grade and grade ≥ 3 infections related to monotherapy mosunetuzumab and odronextumab was not possible due to inconsistent reporting of these outcomes across studies. When used in combination, all-grade infections for epcoritimab were comparable to epcoritimab monotherapy (34% 95%CI: 21 - 47% vs 42% 95%CI: 31 - 52%, z = 1.13, p = 0.258). Mosunetuzumab was given with R-CHOP in a single study, but all-grade and severe infection rates were not reported. This systematic review reports a modest rate of serious infections in lymphoma patients receiving BsAb. The higher rate of fatal viral and fungal infections highlights a potential need for increased clinical vigilance for infections classically associated with T-cell depletion. Although further data is needed, CD20-targeted BsAb may confer higher infection risk than CD19-directed products.

Pharmacodynamic Biomarkers and CtDNA Support the Mechanism of Action and Clinical Efficacy of Golcadomide (CC-99282) Combined with R-CHOP in Previously Untreated Aggressive B-Cell Lymphoma

Introduction Golcadomide (GOLCA, CC-99282) is an orally available cereblon E3 ligase modulator (CELMoD ®) that potently degrades target proteins Ikaros and Aiolos, enhancing antiproliferative, apoptotic, and immunomodulatory activity and preferentially distributing to lymphoid organs. In a phase 1 study, GOLCA monotherapy previously demonstrated a manageable safety profile with promising clinical activity in relapsed/refractory non-Hodgkin lymphoma (Michot Blood 2021). In the ongoing CC-220-DLBCL-001 study (NCT04884035), GOLCA dose finding is underway in patients with previously untreated aggressive B-cell lymphoma (BCL), in combination with R-CHOP in 21-day (D) cycles (C), at a variety of GOLCA dose levels (DLs) and schedules: DL-1 = 0.2 mg D1-7, DL1 = 0.4 mg D1-7, and DL2 = 0.4 mg D1-10. Here, we report Ikaros degradation, immune modulation, circulating tumor DNA (ctDNA) assessment, and baseline tumor genomics. Methods Peripheral blood was collected to analyze Ikaros and Aiolos levels in T cells by flow cytometry (C1D1, C1D7, and C2D1). Immune phenotyping of T and natural killer (NK) cells was performed by flow cytometry (C1D1, C1D15, C2D1, and C3D1). ctDNA levels were analyzed by the PhasED-Seq assay (timepoints C1D1, C2D1, C3D1, and end of treatment [EOT]; Kurtz Nat Biotechnol 2021); somatic variants were filtered by performing PhasED-seq on buffy coats. Pretreatment biopsies were analyzed by whole genome sequencing and whole transcriptome sequencing. DL1 (0.4 mg D1-7) and DL2 (0.4 mg D1-10) data were pooled. Results Ikaros levels in T cells at 5 hours after the first dose had a 1.1% median increase with 0.2 mg and 44% median decrease with 0.4 mg; by C1D7 pretreatment, Ikaros substantially decreased with both DLs (median: 0.2 mg, 87% decrease; 0.4 mg, 83% decrease). Ikaros differences between 0.2 mg and 0.4 mg were not substantial. By C2D1, Ikaros levels recovered to baseline. Based on preclinical benchmarks, steady-state Ikaros degradation in T cells suggested that direct tumor cell killing by GOLCA was achieved. Changes in T and NK cell phenotypes in peripheral blood included activation, increased proliferation, increased T effector memory and regulatory cell levels, and decrease in naive T cells. Changes were seen by C1D15 and maintained at C2D1 and C3D1. Overall, immune changes between 0.2 mg and 0.4 mg dose levels were not substantially different. Phased variant data from ctDNA and buffy coats were evaluable for 34 patients (0.2 mg, n =15; 0.4 mg, n = 19). Baseline ctDNA levels were similar between the 0.2 mg and 0.4 mg cohorts and, consistent with prior studies, correlated with International Prognostic Index and cell of origin. Previously reported landmarks such as early molecular response (EMR; ≥ 2 log decrease) at C2D1 and major molecular response (MMR; ≥ 2.5 log decrease) at C3D1 were assessed (Kurtz J Clin Oncol 2018) . A high fraction of patients designated high risk by pretreatment ctDNA (≥ 2.5 log haploid genome equivalents [HGE]/mL; Kurtz J Clin Oncol 2018) were clinical responders. Patients who achieved minimal residual disease (MRD) negativity on treatment generally remained MRD negative at subsequent timepoints. At C2D1, the EMR and MRD negativity rates for 0.2 mg were 73% (11/15) and 20% (3/15), respectively; for 0.4 mg these rates were 83% (15/18) and 44% (8/18). At C3D1, the MMR and MRD negativity rates for 0.2 mg were 83% (10/12) and 42% (5/12), respectively; for 0.4 mg these were 83% (10/12) and 58% (7/12). Although ctDNA differences between 0.2 mg and 0.4 mg were not substantial, numerically greater ctDNA changes were seen with 0.4 mg GOLCA + R-CHOP vs 0.2 mg GOLCA + R-CHOP and vs R-CHOP and R-CHOP-like regimens from other cohorts ( Figure). Insufficient samples were available to draw conclusions about EOT, but additional ctDNA data will be presented. Whole genome and transcriptome sequencing data from GOLCA + R-CHOP-treated patients will be shown. Conclusions Pharmacodynamic biomarkers and ctDNA levels indicate that both tumor-intrinsic and immune-modulating mechanisms of action of GOLCA remain relevant in combination with R-CHOP in previously untreated aggressive BCL. ctDNA data during treatment are consistent with the efficacy noted on clinical restaging. Translational data demonstrate the potency of GOLCA and will support future frontline clinical trial designs in patients with untreated aggressive BCL. Study support: Celgene, a Bristol-Myers Squibb Company

Digital PCR Assay for Evaluation of CD19-CAR-T Cells In Vivo Expansion in Aggressive B-Cell Lymphoma: Impact on Clinical Outcome

Background: Chimeric antigen receptor (CAR)-T cells therapy has revolutionized hematological treatment mainly in non-Hodgkin lymphoma (NHL) demonstrating unprecedented clinical responses when directed against CD19 combining the antigen-binding properties of antibodies with the effector functions of T cells. In particular, CAR-T cell peak concentrations and area under the curve in the first 28 days after Axicabtagene Ciloleucel (Axi-cel) infusion have been associated with long-term efficacy. Based on a digital PCR assay for sensitive CAR-T cells detection, we aimed to detect the CAR-T product in patients (pts) and associate its kinetics with patient outcome. Methods: A retrospective analysis was conducted involving 28 patients diagnosed with NHL treated with Axi-cel CAR-T products (2019-2023) in the Department of Hematology of the Clinical University Hospital of Valencia, Spain according to the Declaration of Helsinki ethical criteria. 97 peripheral blood mononuclear cell (PBMC) DNA samples were extracted from 3-12 ml EDTA tubes. We performed serial measurements of CAR-T-cell blood levels (7,14, 28 days post-infusion) using a newly developed axi-cel-specific dPCR assay (Bio-Rad,USA) with QIAcuity Digital PCR System and QIAcuitySoftware for data analysis (QIAGEN, Germany). The peak CAR-T-cell blood concentrations (CAR-T-Cmax) and the cumulative CAR-T-cell levels over the first 28 days in PB by area under the curve (AUC CAR-T) were calculated based on CAR-T dPCR values (copies/μL). Response assessment was performed per institutional practice and based on Lugano Criteria. Statistical analyses were performed using SPSS Software (IBM, Germany, v.22). Logistic regression was used to evaluate the relationship between covariate and outcome. Kruskal-Wallis and Dunn's tests were used when 3 subgroups were involved. Survival analyses were conducted according to CAR-T dPCR values. Results: A total of 28 pts (16 [57.1%] females) who received CAR-T cell therapy were included [25 (89%) DLBCL; 3 (11%) PMBCL]. The patient's best overall response assignment (partial or complete) were observed in twenty-two (78,4%) named as responders (R), otherwise six (21,4%) nonresponders (NR) around day 30 of evaluation. CAR-T-CMax ranged from 2.30 to 176.10 (median, 40.16) CAR-T copies/μL, 7 days later of majority expansion day post infusion: R pts had a higher peak expansion of 66.38 copies/µL compared to 32.43 copies/µL although this difference did not reach statistical significance ( p=018). Expansión was faster in R pts (median 7.64 days pot-infusion) versus NR pts (median 12.83, p=0,10) . Quantitative 7-d CAR-T copies/μL, and AUC value from days 0 to 7, showed statistical significance differences post-infusion between R and NR (52.08±32.48 vs 15.51±20.51, p<0.005; 3603.88±2520.02 vs 1291.74±1948.77, p<0.037). As for response assessment, 7-d CAR-T values were able to successfully predict the development of NR pts with an area under the curve of 0.841 (CI 95% 0.66-1.00), a sensitivity of 0.950 and a specificity of 0.625 (Fig 1.). Eleven pts (39.3%) died at a median of 3.6 months (1.7-6.1 months) post-infusion. For survival analyses, a cut-off value of 18.35 7-d CAR-T copies/μL that maximized the Log-Rank statistic, was selected to divide pts into R and NR categories. Median overall survival (OS) was 23.9 [16.3-NA, 95% CI] months and 13.2 [3.30-3.96, 95% CI] months for R and NR according to CAR-T copies/μL 7 days, respectively (Fig.2). Conclusions: Digital PCR for CAR-T evaluation shows that quantitative CAR-T copies/μL could be a potential predictor of short-term response and survival at first 7 days post-infusion value in pts with DLBCL or PMBCL treated with CAR-T cell therapy.

Evaluation of the T-Cell Metabolic State of Starting Material and Manufactured CAR T Cell Products and Clinical Outcome of CAR T-Cell Therapy for Large B Cell Lymphoma

Background Although chimeric antigen receptor (CAR)-T cells are a viable treatment option for patients with relapsed or refractory (r/r) aggressive B-cell lymphomas, more than half of patients experience relapse within the first two years after product infusion. Successful autologous cell-based anti-cancer therapies require persistence of effector cells, features that highly depend on complex metabolic processes. We earlier demonstrated in chronic lymphocytic leukemia, that expansion and persistence of CD19-CAR T-cell infusion in vivo was correlated with increased mitochondrial mass and enhanced biogenesis(van Bruggen et al, Blood 2019). Metabolic features might therefore serve as early biomarker tools to predict efficacy and outcome of CAR T-cell therapy and might allow therapeutic targeting but did was never studied in a prospective clinical trial. Aims Perform extensive phenotyping and state-of-the-art metabolic state analyses of T cells prior and post CAR T cell production and investigate correlations with clinical outcomes and patient characteristics. Methods Samples were obtained from large B cell lymphoma (LBCL) patients that were part of the TRANSCEND-NHL-001 trial (NCT02631044, Abramson et al, Lancet 2020).Thirty-two patients with r/r B-cell lymphoma who received CAR-T cell treatment lisocabtagene maraleucel (16 responders (R) and 16 non-responders (NR)) were included in our analyses and (metabolically) analyzed by flow cytometry and extracellular flux analysis. Metabolic phenotyping was performed on 53 samples at baseline and 44 samples after an in vitro stimulation. Extracellular flux analysis was done on 14 DP samples. The cohort was split evenly between Responders and Non-responders, and balanced for a score calculated taking key clinical baseline characteristics that were found to be associated with response into account (NCT02631044, Abramson et al, Lancet 2020). Both T cells obtained at time of leukopheresis (starting material (SMAT)) and CAR-T drug products (DP), (following transduction of a CD19-targeted CAR T cell product with a defined CD4 and CD8 composition bearing a 4-1BB costimulatory domain and expansion) were analyzed directly after thawing and after stimulation with anti-CAR-antibody in case of CDP, or αCD3/αCD28 antibodies for SMAT samples. Patients consented to these studies and researchers were blinded for clinical outcomes at time of analysis. Results We found that the baseline clinical factors that associate with clinical response are related to specific metabolic cellular phenotypes, both in SMAT and DP, and with and without T cell stimulation. SMAT samples from patients with higher baseline scores show signs of an increased exhausted phenotype with regards to cellular ROS and exhaustion marker expression, whilst a more plentiful memory-like phenotype was seen in patients with a lower baseline score. When clustering by clinical response status, analysis of glucose and fatty-acid uptake, neutral lipid content, cellular ROS, mitochondrial mass and potential and flux data showed that responders, in both SMAT and DP samples, have an all-round increased metabolic activity upon αCD3/αCD28 or CAR T-receptor triggering, respectively, when compared to non-responders (Figure 1A). Furthermore, responding patients show enrichment of CD27 expression in the SMAT material, indicating a more abundant memory phenotype. Summary/Conclusions This is the first evidence of an association between a specific immune-metabolic phenotype of (CAR-) T cells and outcome of CAR-T cell therapy in patients with relapsed/refractory LBCL. More specifically, a higher abundance of memory phenotypes, metabolic activity and mitochondrial biogenesis of both harvested T cells and CAR-T product associates with improved clinical response. Combining metabolic, phenotypical and clinical characteristics and biomarkers of patient T cells (both pre- or post-production) shows the importance of the metabolic phenotype and fitness of T cell starting material, underlying the importance of early line CAR T therapy following diagnosis.

Molecular Mechanism of Action of Glucocorticoids in Lymphoma Therapy

Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas, including diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). GCs act by binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. The transcriptional response to GCs can vary significantly due to the variation in expression levels of GR cofactors and chromatin landscapes in different cell types. The mechanisms by which GCs kill malignant lymphoma cells are largely unknown, prompting us to search for new targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To identify genes that synergize or antagonize GC lethality in lymphomas, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone, the active metabolite of prednisone. Screens in cell line models of BL and DLBCL (both ABC and GCB subtypes) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor signaling pathway, which is required to sustain the viability of these malignant lymphoma cells. With combination of the cleavage under targets and release using nuclease (CUT&RUN) assay and RNA-seq, we identified GR binding to the LAPTM5 locus at its glucocorticoid response element and AP1 motifs upon GC treatment. GR binding also induced expression of LAPTM5, which negatively regulated BCR signaling by promoting the lysosomal degradation of the BCR. Conversely, GC induced binding of GR to the CSK locus, thereby repressing expression of the non-receptor tyrosine kinase CSK, which antagonizes BCR signaling by phosphorylating an inhibitory tyrosine residue present in all Src-family kinases (SFKs). However, in BCR-dependent aggressive lymphomas, inactivation of CSK paradoxically decreased proximal BCR signaling and induced cell death. By performing quantitative phosphoproteome and ubiquitinome with mass spectrometry, we demonstrated that treatment of lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi) initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-kB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs, which have been used empirically for decades to treat lymphomas. GCs restrain the most proximal steps in BCR signaling at the plasma membrane by, on one hand, decreasing BCR abundance, and on the other hand, by decreasing CSK expression, thereby reducing expression of the essential SFKs. Small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers.

A Single Cell Atlas of Diffuse Large B Cell Lymphomas Reveals Distinct Cellular States Predictive of Outcomes

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell lymphoma with high clinical and biological heterogeneity. Despite current effective immunochemotherapy, up to 40% of patients do not respond or develop refractory disease. DLBCL is characterized by two major cell-of-origin (COO) subtypes, germinal center B cell-like (GCB) and activated B cell-like (ABC), with recent research uncovering additional molecular subgroups based on genomic alterations or tumor microenvironment (TME) features. However, current classifiers use bulk or deconvolution profiling which blurs intra-tumor heterogeneity, hindering the discovery of true functional cell states underlying clinical diversity. Here, we constructed a multimodal single-cell atlas of 62 DLBCL cases, integrating single-cell transcriptomes, immune repertoire and genetic features, to decrypt recurrent B cell states and TME ecosystems while identifying clinically relevant prognostic biomarkers (Fig.1). We performed simultaneous 5'-end single-cell RNA, B-cell receptor (BCR) and T-cell receptor sequencing on 63 DLBCL lymph node biopsies, recovering 109,294 cells after stringent quality controls. These biopsies were obtained from the real-world, clinically-annotated, multicentric CeVi collection and included 43 patient samples at diagnosis (ABC = 17, GCB = 11, Unclassified = 6, Not Evaluated = 9; 21 of which did not achieve event-free survival at 24 months (EFS24) after R-chemotherapy regimens), 11 relapsed/refractory DLBCL and 8 DLBCL transformed from indolent lymphoma. We distinguished malignant B-cells from TME using canonical markers and BCR sequence, exploring heterogeneity in both components. We developed an original bioinformatic approach to define malignant B cell transcriptional archetypes shared across patients. To assess whether subclonal genetics are associated with transcriptional states, we inferred copy number variations (CNV) and studied BCR intraclonal variations of malignant cells. We also integrated TME data (39,674 cells) to quantify subpopulations and clonally expanded T-cell subsets, and performed robust statistical analyses to identify clinically relevant features linked to patient outcomes. To circumvent interpatient heterogeneity in malignant B cells, we defined transcriptional cellular archetypes conserved across patients by combining 3 unsupervised methods: rank-biased overlap meta-clustering, variational autoencoder and canonical correlation analysis integration. We identified 5 cellular archetypes (Arch.1-5): Arch.1 was characterized by plasma cell identity, Arch.2 by MYC and NFKB pathways, Arch.3 by MYC pathway only, Arch.4 by quiescence markers, and Arch.5 by GC markers. Several archetypes co-existed in each tumor sample, and archetype identity was superimposed to the transcriptional profile conferred by COO molecular class as an additional layer of transcriptional heterogeneity. Cell state-specific CNV and BCR subclones were very rare, suggesting that subclonal genetic changes were not the main drivers of malignant B cell plasticity and archetypes. Kaplan-Meier analysis showed a significant negative association between proportion of Arch.4 cells and continuous EFS in our diagnosis cohort. Unsupervised analysis of archetype composition further distinguished ABC patients who achieved EFS24 (enriched in Arch.3 cells) from those with a particularly poor prognosis (enriched in Arch.4 cells). Integrated analysis of the TME revealed three major ecosystems, including one enriched for effector and cytotoxic T cells. In diagnosis samples, pseudo-temporal trajectory analysis revealed the enrichment of clonally-expanded hyperactivated CD8+ T cells expressing canonical exhaustion markers in patients achieving EFS24, suggesting a positive link between the extent of CD8+ T cell differentiation and response to R-CHOP based regimens. Shedding new light on DLBCL biological and clinical heterogeneity, our comprehensive single-cell atlas of DLBCL uncovered recurrent tumor cell states and TME features crucial for stratifying non-responsive ABC patients and guiding treatment decisions. Ongoing single-cell spatial transcriptomics analyses will be used to further validate our findings and elucidate archetype-TME interactions that may drive malignant B cell plasticity, with the potential to uncover novel therapeutic targets.

The KIR-HLA-CD16a Immunogenetic Profile Influences NK Cell-Mediated ADCC and Response to Rituximab Therapy in B-Cell Lymphomas

Rituximab-based chemoimmunotherapy (R-CHOP) is the standard of care in most B-cell lymphomas, however there is marked interpatient heterogeneity in treatment response. An important mechanism of Rituximab (anti-CD20) action is through natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but the factors influencing this are poorly understood. Here, we present findings from a preclinical study investigating the role of NK cell receptor polymorphisms and HLA allelic variation on ADCC against aggressive B-cell lymphoma cell lines. Genotyping was conducted using Luminex-based SSO and Sanger sequencing. Multicolor flow cytometry was used to assess the response of activated NK cells from healthy donors against 8 lymphoma cell lines. Individuals carrying the GG or GT allele in CD16a were found to have a 3-8 fold greater NK cell response compared to individuals with a TT allele and rituximab-mediated ADCC was significantly greater in individuals with a KIR-3DL1 allele when tested against B-cell lymphomas lacking the HLA-Bw4 allele. An additive effect was observed when considering both CD16a and KIR-HLA together. As expected, CD20 expression was a strong correlate of NK cell response. In summary, NK cell-mediated ADCC in response to ADCC appears to be influenced by the KIR-HLA-CD16a genotype. These findings suggest a potential role for immunogenotyping in B-cell lymphomas treated with Rituximab. Additionally, the presence of the shared F c receptor domain opens the possibility of applying this framework towards predicting NK cell responses to antibody therapies in other diseases.

Patterns of Care and Prognostic Modeling Following Follicular Lymphoma Transformation to Aggressive B-Cell Lymphoma: An Analysis from the LEO Consortium

Introduction: Histologic transformation (HT) from follicular lymphoma (FL) to an aggressive lymphoma is infrequent but has unfavorable outcomes. Sarkozy et al. (J Clin Oncol, 2019) reported that approximately half of FL-related deaths within the first decade of diagnosis occurred following HT. Our recent analysis showed heterogeneous outcomes with median overall survival (OS) of 61 months (Day et al., ASH, 2022). Prognostic tools post HT would be clinically useful but development has been limited by small cohort sizes. Factors previously associated with outcomes following HT have included age, time to progression, prior therapy, anthracycline exposure, and early HT. Utilizing the Lymphoma Epidemiology of Outcomes (LEO) Consortium of Real World Evidence (CReWE), we describe patterns of care and evaluate other prognostic factors following HT. Methods: This multicenter observational cohort consists of prospectively enrolled patients in the LEO Cohort, and patients with HT retrospectively identified at LEO centers. Patients diagnosed with biopsy proven grade 1-3A FL at diagnosis, and a subsequent biopsy showing HT to an aggressive lymphoma (FL grade 3B, diffuse large B cell lymphoma [DLBCL], or high-grade B cell lymphoma [HGBCL]) between 2002-2022, were eligible for the study. Patients with a component of FL grade 3B, DLBCL, or HGBCL at diagnosis were excluded. HGBCL was defined as double or triple hit or another aggressive subtype. OS was defined as the time from HT until death from any cause and calculated via Kaplan-Meier. Associations between clinical factors at time of HT and/or diagnosis and OS were evaluated using Cox models. Cause of death was evaluated using a competing risk approach. Results: Overall, 328 patients were confirmed with HT during the study period, 41% were female, and the median age at HT was 63 years (interquartile range [IQR] 56-70). The median time from diagnosis to transformation (TDT) was 35 months (IQR 12-66). At HT, 88% had advanced stage disease (III-IV) and LDH was elevated in 49%, while few patients exhibited B symptoms (5%) and ECOG performance status was >2 in 9%. 23% did not receive systemic therapy prior to HT. For those with prior therapy, the median time from prior therapy to HT was 15 months (IQR 7-39). The most frequent treatment regimens following HT were CD20-CHOP (42%), followed by platinum-based salvage therapy (20%), R-EPOCH (10%), CD20-Benda (8%), R-Mono (4%), and R-Gem-Ox (3%). 50 (15%) patients underwent consolidation with autologous stem cell transplant, 3 underwent allogeneic transplant (30 platinum-based, 15 CD20-CHOP, 5 R-EPOCH, 3 Other), and 2 received CAR T-cell therapy (Figure 1). After median follow-up of 85 months from HT, 170 patients had died; 5-year OS was 52% and median OS was 65 months (95% CI: 55-107). Among 160 patient deaths, cause was most often lymphoma-related (progression, n=107 [63%]; therapy-related, n=14 [7%]) compared to non-lymphoma (n=20, 13%) or missing/unknown causes (n=29, 17%). Clinical factors at the time of HT that were associated with worse OS include: ECOG PS >2, age ≥70, IPI 3-5, and shorter TDT (<24 months), hemoglobin <10 g/dl and elevated LDH. A transformation to HGBCL was also associated with inferior OS (Table 1). OS did not significantly differ by gender, BMI, stage, presence of B-symptoms, absolute lymphocyte count, number of extranodal groups, presence of bulky disease at HT, or response to prior FL treatment. Factors at the time of FL diagnosis that were associated with decreased OS after HT included FLIPI score 3-5 (high risk), FLIPI 2 (moderate risk), and FL grade 3A at diagnosis. Exposure to any systemic therapy for FL prior to HT was associated with shorter OS, with the worst outcomes in patients receiving an anthracycline prior to HT. POD24 to frontline immunochemotherapy was associated with inferior OS after HT (Table 1). Conclusions: Patterns of care in Transformed FL are heterogeneous and vary greatly in intensity. These data confirm previous reports that age, early HT, and any systemic therapies prior to HT are negative prognostic indicators for OS. At HT elevated LDH, low hemoglobin, POD24 status, poor performance status, and HT to HGBCL were associated with poor outcomes following HT. These data provide a basis for future prognostic indices to identify patients with highest risk of lymphoma related mortality for whom study of novel treatments is greatly needed.

Baseline Immunoglobulin G and Immune Function in Lymphoma: A Retrospective Analysis

Introduction: Non-Hodgkin's lymphoma (NHL) encompasses a diverse group of lymphoma subtypes with a wide range in disease course. Previous studies show that hypogammaglobulinemia in treatment-naïve patients is associated with poorer survival in high grade B-cell non-Hodgkin's lymphomas, though data is lacking on how this applies across all B-cell lymphoid malignancies. We conducted a retrospective study of immunoglobulin levels and clinical outcomes including survival, hospitalization, and infection rates in patients diagnosed with B-cell non-Hodgkin lymphomas of all grades at our institution. Methods: Patient data was collected from January 2010 to June 2022. Two-hundred twenty-three adults (aged > 18 years) with available pre-treatment IgG levels were selected, with hypogammaglobulinemia defined as IgG< 500 mg/mL. For this analysis, DLBCL (n=90), Primary CNS (n=5), and Burkitt's lymphoma (n=1) was classified as high-grade, while CLL (n=52), mantle cell (n=20), marginal zone (n=25), follicular (n=21), and lymphoplasmacytic lymphomas (n=5) as low-grade. Baseline clinical, staging, and laboratory information was collected including hospital admissions, date of death, and number of infections. Preliminary results for overall survival, 3-year survival and 5-year survival were also analyzed. Multivariate logistic models were used to predict outcomes dichotomized for baseline IGG and high/low grade cancers. Kaplan-Meier estimates and Cox model were performed to analyze the overall survival data. Results: The incidence of pre-treatment hypogammaglobulinemia was 47% in the high-grade B-cell lymphoma cohort and 57% the low-grade B-cell lymphoma cohort (p=.9371). Across all NHL subtypes, individuals with baseline IgG< 500 showed an increased rate of hospitalization (4.453, CI: 1.955-10.54, p= 0.0005) and higher mortality (3.325, CI:1.258, 8.491, p= 0.013), yet no association in number of infections when compared with those with IgG>500. There was no statistical difference in patients' hospitalization rate when they were treated with IVIG only, rituximab only, or a combination (p=.141792). Additionally, there was no statistically significant correlation with COVID infections when evaluating grade of lymphoma (p=.8527) or IgG level (p=.6535). There was no statistically significant difference in individuals who were alive after three years and five years in those with baseline IgG<500. Discussion: Our study is the first to analyze incidence of hypogammaglobulinemia at time of diagnosis of NHL as a potential biomarker of interest for future outcomes including hospitalization and infection. These findings demonstrate an association between hypogammaglobulinemia when comparing the rate of hospitalization and mortality across the spectrum of B-cell NHL. This effect was further amplified in patients with aggressive B-cell lymphoma. Given the common use of monoclonal antibodies to treat B cell lymphomas of all subtypes and the known risk of hypogammaglobulinemia from these treatments and the common finding of hypogammaglobulinemia at diagnosis of B cell NHL, pretreatment screening of IgG levels may be important to identify patients at risk for infection, hospitalization and death. Further clinical equipoise exists requiring a randomized clinical trial to indicate the use as a prognostic factor for infection, hospitalization, and mortality.