Wang, Yucai

Abstract Background Although CAR T-cell therapy has achieved remarkable response in patients (pts) with B-cell non-Hodgkin’s lymphoma (NHL), 20-30% of pts still have primary refractory disease (PD1), and another 30-40% relapse (PD2). We and others have shown that monocytes can modulate T-cell functions and impact clinical response to chemoimmunotherapy in NHL. Given that monocytes nadir and recovery kinetics differ from lymphocytes post lymphodepletion chemotherapy, we hypothesize that monocyte phenotype at peak CAR-T expansion and in the early month post CAR-T can modulate immune responses. Methods scRNAseq were performed on peripheral blood mononuclear cells of NHL pts prior to lymphodepletion chemotherapy (pre), at peak CAR-T expansion (peak), and one month following CAR-T (M1). Seurat v4.3.0 was used for unsupervised hierarchical clustering analysis. Lugano criteria was used to determine clinical responses which were categorized as CR (complete remission), PD1, or PD2. Results We analyzed 129,981 monocytes from 77 samples of 32 pts (16 CR, 4 PD1, 12 PD2) and 5 healthy controls. Unsupervised hierarchical clustering analysis identified 13 monocyte clusters: 4 classical monocyte (CM) clusters with transcriptome profiles for enriched signaling in IL-6, TGFβ, EMT, and p53; 2 intermediate monocyte (IM) clusters enriched for IL-1 or CD38 signaling; 2 non-classical monocytes (NM), one with increased CD36 expression; 3 monocytic MDSC clusters with enrichment of TGFβ, SIRPα, and HIF1α signaling; and 2 lymphoma-associated monocytes, expressing CD163 or CD169. At Pre and Peak, the distribution of the monocyte clusters was similar in pts with CR, PD2, or PD1, with only a slight decrease in the percent of NM in the PD2 group at Pre and slight increase of CD36 NM in PD1 at Peak. Compared to Pre, all pts had increased percent of CD36 NM and IL-1 IM at Peak. Interestingly, while the distribution of monocyte clusters was similar at Pre and Peak across clinical responses, GSEA showed that monocytes in CR pts, compared to PD1 and PD2, had gene expression enrichment of MYC target V2 pathway at Pre and TGFβ pathway at Peak. At M1, pts in PD1 had increased percent of p53 and TGFβ CM and decreased percent of NM. In contrast, pts with CR had decreased percent of IL-6 CM, p53 CM, TGFβ CM, IL-1 IM and increased percent of NM. Notably, as pts with CR and PD2 may appear similar clinically at M1, pts with PD2, compared to CR, have increased IL-6 CM, IL-1 IM, and decreased NM. In addition, GSEA showed that PD2 pts had increased IFNα response. Our results demonstrate that monocyte phenotype differences at Pre and Peak could be associated with CAR-T clinical response, and that persistent inflammatory monocyte signaling at M1 in pts with clinical response is associated with relapse. These data suggest a role for development of monocyte biomarkers to predict response and therapeutic targeting. Citation Format: Andre de Menezes Silva Corraes, Panwen Wang, Radhika Bansal, 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 Castro, Saad Kenderian, Haidong Dong, Ying Li, Yi Lin. Monocyte transcriptome profiles associated with clinical response in patients with B-cell non-Hodgkin’s lymphoma receiving CD19-directed 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 4001.

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 Background: The impact of a prior history of non-hematologic malignancy (NHM) on clinical outcomes among individuals with newly diagnosed chronic lymphocytic leukemia (CLL) or monoclonal B-cell lymphocytosis (MBL), the precursor to CLL, is not well known. Methods: In this prospective study, we identified individuals with CLL/MBL who were seen in the Division of Hematology at the Mayo Clinic, Rochester. Participants provided consent within nine months of diagnosis, had no prior history of hematological malignancy, and completed a self-reported risk-factor questionnaire, including a prior history of NHM. Time to first treatment (TTFT) was defined as the time between the date of diagnosis and the earliest date of the first CLL treatment, death, or the last known treatment-free follow-up date. Overall survival (OS) was defined as the time from diagnosis to death or the last follow-up. We used Cox regression to estimate hazard ratios (HR) and 95% confidence intervals (CI) for associations between a prior history of a NHM with OS and TTFT. We adjusted for sex and the CLL International Prognostic Index (CLL-IPI), a score comprised of five key CLL prognostic factors, including age and Rai stage. Results: Among the 1,090 CLL/MBL individuals, 833 (76%) had CLL. The median age at diagnosis was 64 years (range 24-89), with 65% being men, and 230 (21%) reported a prior history of NHM. The most common tumor was non-melanoma skin cancer (NMSC, 8.9%, 96 individuals), followed by prostate, testicular, and penile tumors (7.3%, 51 individuals), and melanoma (1.9%, 20 individuals). CLL/MBL patients with a prior history of a NHM were more likely to be over 65 years old (29%) than under 65 years old (14%, p < 0.0001). After a median follow-up of 2.9 years (range 0-21), 470 CLL/MBL patients progressed, requiring CLL treatment. CLL/MBL individuals with a prior history of NHM had a longer TTFT compared to those without (HR=0.69; 95% CI: 0.53-0.90; p=0.006). Individuals with a prior history of NHM who had either intermediate or high-very high CLL-IPI score had significantly longer TTFT (HR= 0.55; 95% CI: 0.33-0.92; p=0.02, and HR=0.65; 95% CI: 0.43-0.97; p=0.03, respectively) compared to those without NHM. After a median follow-up of 8.6 years (range 0-21), 359 individuals died. CLL/MBL patients with a prior history of NHM also experienced reduced OS compared to those without (HR=1.46; 95% CI: 1.08-1.97, p=0.013), particularly among those with low (HR=1.70; 95% CI: 0.96-3.00, p=0.07) or intermediate-risk (HR=2.08; 95% CI: 1.24-3.51, p=0.006), according to CLL-IPI. After excluding NMSC, results remained constant. Conclusions: At the time of diagnosis, one out of five CLL/MBL individuals had a prior history of a NHM. These individuals had a longer time to first CLL directed treatment and reduced OS, even after accounting for other important CLL prognostic factors, which deserves additional studies. Citation Format: Bryan A. Vallejo, Cristine Allmer, Kari G. Rabe, Dennis P. Robinson, Yucai Wang, Paul J. Hampel, Esteban D. Braggio, Neil E. Kay, James R. Cerhan, Sameer A. Parikh, Susan L. Slager. The impact of prior history of non-hematologic malignancies on time to first treatment and overall survival among individuals with CLL or MBL [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 7450.

Abstract Introduction: Chimeric antigen receptor T-cell (CAR-T) therapy is an established treatment modality for the management of refractory and/or relapsed non-Hodgkin lymphoma (NHL). Despite restrictions on granulocyte colony-stimulating factor (G-CSF) use in registration clinical trials due to potential worsening of cytokine release syndrome (CRS), G-CSF is often prescribed for neutrophil recovery post-CAR-T in clinical practice. We conducted a retrospective study to assess the association between G-CSF use and the safety and efficacy of CAR-T. Methods: Following Institutional Review Board approval, we retrospectively analyzed NHL patients receiving commercial CD19-targeted CAR-T across Mayo Clinic sites (Arizona, Florida, and Minnesota) between October 2017 and May 2023. Demographics, labs, and post CAR-T events were collected. Electronic medical records were reviewed for G-CSF exposure within Day 1 - 100 post-CAR T. Differences by G-CSF status were examined using a Chi-square test and Kruskal Wallis tests. Hazard ratios (HR) and 95% confidence intervals (CI) for overall survival (OS) were calculated using Cox model. Results: Among 270 CAR-T patients; 106 received G-CSF within 30 days and 164 did not. Median age was 56.9 with G-CSF and 58.3 without G-CSF. Most were male (75.2% G-CSF, 62.6% no G-CSF). Diffuse large B cell lymphoma was the predominant NHL subtype (67.0% G-CSF, 65.2% no G-CSF). G-CSF within 30 days was not associated with reduced incidence of neutropenic fever (11.7% G-CSF vs 9.9% no G-CSF, p = 0.6593) or documented infection (22.1% vs 18.3%, p = 0.4439). G-CSF correlated with decreased profound neutropenia duration (ANC < 100 μL, 0 vs 2 days, p < 0.0001) and incidence within 30 days ( 31.7% vs 60.4%, p < 0.0001) as well as incidence of protracted profound neutropenia (> 7 days, 3.8% vs 22.8%, p < 0.0001). G-CSF was associated with a higher incidence of cytopenias between days 30 -100: severe neutropenia (ANC < 500/uL, 28.8% vs 15.2%, p = 0.0075), severe anemia (Hb < 8 g/dL, 21% vs 9.7%, p = 0.0114), and severe thrombocytopenia (Plt < 50 g/L, 35.2% vs 20.9%, p = 0.0099). Logistic regression analysis showed that none of the examined covariates, including prior autologous stem cell transplant, ECOG PS score, and receipt of bridging therapy, were associated with severe neutropenia likelihood. G-CSF was associated with a higher CRS incidence (89.6% vs 79.9%, p = 0.0343), but grade 3 CRS or higher (4.7% vs 3.7%, p = 0.6675) was no different. No difference in complete response rates (66% vs 61.1%, p = 0.4707) or 2-year OS (p = 0.523) was observed between the 2 groups. Conclusion: G-CSF within 30 days was associated with reduced duration and incidence of profound neutropenia without a significant impact on incidence of neutropenic fever or documented infections. There was a signal of association between G-CSF and increased incidence of low-grade CRS and delayed cytopenias (day 30-100) that warrants confirmation in larger studies. Citation Format: Lay She Ng, Claire I. Yee, Radhika Bansal, Mohamed A. Kharfan-Dabaja, Grzegorz S. Nowakowski, Allison C. Rosenthal, Javier Munoz, Januario Castro, Wern Lynn Ng, Hemant S. Murthy, Yi Lin, Matthew R. Buras, Madiha Iqbal, Yucai Wang, Talal Hilal. Impact of G-CSF on safety and efficacy of CAR T-cell therapy in non-Hodgkin lymphoma [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 6327.

PURPOSE Limited evidence exists regarding methotrexate (MTX) resumption after patients with lymphoma receive glucarpidase for toxic MTX levels and acute kidney injury (AKI). METHODS This retrospective review included adults with lymphoma treated with glucarpidase after MTX at Mayo Clinic between January 31, 2020, and October 10, 2022. Descriptive statistics summarize patient characteristics and clinical outcomes. RESULTS Of 11 patients treated with glucarpidase after MTX, seven (64%) were rechallenged with MTX. Indications for MTX rechallenge included confirmed CNS disease (n = 6, 86%) and intravascular lymphoma (n = 1, 14%). Compared with the nonrechallenged subgroup, before receiving MTX that required glucarpidase rescue, the rechallenged patients had lower median pretreatment serum creatinine (Scr; 0.7 v 1.2 mg/dL), and none had AKI with previous MTX doses, n = 0 (0%) versus n = 2 (50%). During the MTX dose requiring glucarpidase rescue, the rechallenged group had lower median peak Scr (1.26 v 3.32 mg/dL) and lower incidence of AKI stage III (n = 1 [14%] v n = 3 [75%]), and none of the rechallenged patients required renal replacement therapy (RRT; n = 0 [0%] v n = 1 [25%]). At the first rechallenge after glucarpidase administration, the median MTX dose reduction was 56% (range, 46%-75%), and the lowest used dose when prescribed according to each treatment protocol schedule was 1.5 g/m2. Two (29%) patients experienced AKI (n = 1 stage I, n = 1 stage II) after MTX rechallenge. Zero patients required RRT, and zero required another glucarpidase administration. Six (86%) patients completed all recommended MTX doses. CONCLUSION In selected adults with lymphoma who required glucarpidase for toxic MTX levels after administration of high-dose MTX, resumption of MTX therapy at lower doses is safe. Patients selected for MTX resumption had experienced less severe AKI during the previous cycle compared with those not selected for MTX resumption.

AbstractTo address the current and long‐term unmet health needs of the growing population of non‐Hodgkin lymphoma (NHL) patients, we established the Lymphoma Epidemiology of Outcomes (LEO) cohort study (NCT02736357; https://leocohort.org/). A total of 7735 newly diagnosed patients aged 18 years and older with NHL were prospectively enrolled from 7/1/2015 to 5/31/2020 at 8 academic centers in the United States. The median age at diagnosis was 62 years (range, 18–99). Participants came from 49 US states and included 538 Black/African‐Americans (AA), 822 Hispanics (regardless of race), 3386 women, 716 age <40 years, and 1513 rural residents. At study baseline, we abstracted clinical, pathology, and treatment data; banked serum/plasma (N = 5883, 76.0%) and germline DNA (N = 5465, 70.7%); constructed tissue microarrays for four major NHL subtypes (N = 1189); and collected quality of life (N = 5281, 68.3%) and epidemiologic risk factor (N = 4489, 58.0%) data. Through August 2022, there were 1492 deaths. Compared to population‐based SEER data (2015–2019), LEO participants had a similar distribution of gender, AA race, Hispanic ethnicity, and NHL subtype, while LEO was underrepresented for patients who were Asian and aged 80 years and above. Observed overall survival rates for LEO at 1 and 2 years were similar to population‐based SEER rates for indolent B‐cell (follicular and marginal zone) and T‐cell lymphomas, but were 10%–15% higher than SEER rates for aggressive B‐cell subtypes (diffuse large B‐cell and mantle cell). The LEO cohort is a robust and comprehensive national resource to address the role of clinical, tumor, host genetic, epidemiologic, and other biologic factors in NHL prognosis and survivorship.

Abstract Cytopenias represent the most common side effect of CAR T-cell therapy (CAR-T) and can predispose for severe infectious complications. Current grading systems, such as the Common Terminology Criteria for Adverse Events (CTCAE), neither reflect the unique quality of post–CAR-T neutrophil recovery, nor do they reflect the inherent risk of infections due to protracted neutropenia. For this reason, a novel EHA/EBMT consensus grading was recently developed for Immune Effector Cell-Associated HematoToxicity (ICAHT). In this multicenter, observational study, we applied the grading system to a large real-world cohort of 549 patients treated with BCMA- or CD19-directed CAR-T for refractory B-cell malignancies (112 multiple myeloma [MM], 334 large B-cell lymphoma [LBCL], 103 mantle cell lymphoma [MCL]) and examined the clinical sequelae of severe (≥3°) ICAHT. The ICAHT grading was strongly associated with the cumulative duration of severe neutropenia (r = 0.92, P &lt; .0001), the presence of multilineage cytopenias, and the use of platelet and red blood cell transfusions. We noted an increased rate of severe ICAHT in patients with MCL vs those with LBCL and MM (28% vs 23% vs 15%). Severe ICAHT was associated with a higher rate of severe infections (49% vs 13%, P &lt; .0001), increased nonrelapse mortality (14% vs 4%, P &lt; .0001), and inferior survival outcomes (1-year progression-free survival: 35% vs 51%, 1-year overall survival: 52% vs 73%, both P &lt; .0001). Importantly, the ICAHT grading demonstrated superior capacity to predict severe infections compared with the CTCAE grading (c-index 0.73 vs 0.55, P &lt; .0001 vs nonsignificant). Taken together, these data highlight the clinical relevance of the novel grading system and support the reporting of ICAHT severity in clinical trials evaluating CAR-T therapies.

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.

Background Primary mediastinal large B-cell lymphoma (PMBCL) is an uncommon type of non-Hodgkin lymphoma. Clinicopathologic features are distinct from diffuse large B cell lymphoma, but it shares some clinical and biologic features with Nodular sclerosis classic Hodgkin lymphoma. Central nervous system (CNS) relapse appears to be rare in patients with PMBCL, with limited literature reporting a rate of approximately 1.5% to 4%. We examined the incidence of CNS relapse, treatment approaches and survival outcomes in patients with PMBCL. Methods Patients with newly diagnosed PMBCL seen at Mayo Clinic between 2002 and 2022 were identified using the prospective Mayo Clinic Lymphoma Database. Clinical features, treatment details and outcomes were abstracted from the medical record. Cumulative incidence of CNS relapse was analyzed with death as a competing risk. The association of CNS-IPI, systemic treatment, and CNS prophylaxis approach with CNS relapse rate was analyzed using competing risk models and descriptively. Event-free survival (EFS) for the cohort was defined as time from PMBCL diagnosis to first recorded CNS and/or systemic relapse or death from any cause. Overall survival (OS) was defined as time from PMBCL diagnosis (or CNS relapse) to death from any cause. Survival analysis was performed using the Kaplan-Meier method. Results A total of 124 PMBCL cases were identified. The median age at diagnosis was 37 years and 61% were female. The distribution of CNS-IPI was low-risk in 67 (61%), intermediate-risk in 41 (37%), and high-risk in 2 (2%). 64 patients (52%) received R-CHOP and 54 patients (44%) received R-DA-EPOCH as frontline therapy. High-dose methotrexate (HD-MTX) for CNS prophylaxis was administered to 6 patients (5%), 5 with R-CHOP and 1 with R-DA-EPOCH. 16 patients received intrathecal (IT) chemotherapy (methotrexate and/or cytarabine) for CNS prophylaxis, 3 with R-CHOP and 13 with R-DA-EPOCH. The median follow-up was 56.0 months. The 5-year EFS rate was 75.1% and the 5-year OS rate was 90.7% for the entire cohort. 20 patients had systemic relapse only and 3 had CNS relapse. The cumulative incidence of CNS relapse was 1.84% (95% CI 0.46%-7.17%) at 2 years and 2.92% (95% CI 0.50%-8.84%) at 5 years (Figure 1). No statistically significant association of CNS-IPI, systemic therapy or CNS prophylaxis regimen with CNS relapse rate was found in competing risk models; however, this analysis was limited by a small event number (n=3). Among the 3 patients with CNS relapse, 1 had high-risk CNS-IPI and 2 had intermediate risk. All 3 patients were treated initially with R-CHOP. Two patients did not receive any CNS prophylaxis and 1 patient received IT CNS prophylaxis. All three patients who had CNS relapse were female. Patient #1 was diagnosed at age 48 and had simultaneous CNS and systemic relapse 6 months later. The patient was treated with HD-MTX, temozolomide and rituximab for CNS relapse, and subsequently received multiple therapies for systemic disease including R-DHAP, R-ICE, and died 17 months after CNS relapse. Patient #2 was diagnosed at age 22 and had CNS relapse after 8.7 months, followed by systemic relapse 2 months later. The patient received HD-MTX, temozolomide and rituximab for CNS relapse, followed by systemic and whole-brain radiation therapy (RT) for both systemic and CNS relapse. Subsequently the patient received salvage chemotherapy, autologous and allogeneic stem cell transplant for systemic relapse. The patient died 26.8 months after CNS relapse. Patient #3 was diagnosed at age 24 and had systemic relapse after 20.7 months, followed by CNS relapse 5.4 months later. The patient received methotrexate and whole-brain RT following CNS relapse and died 1.2 months after CNS relapse. Besides these 3 patients, we identified 6 PMBCL patients with CNS relapse who were diagnosed at other medical centers and subsequently referred to our institution for management of refractory disease. The survival after CNS relapse was poor, with a median OS of 10.4 months (95% CI 5.6-15.2). Conclusion Our study showed that the CNS relapse rate (1.84% at 2 years and 2.92% at 5 years) was overall low in patients with PMBCL, consistent with previous reports. CNS relapse was associated with poor survival despite aggressive treatment approaches. Novel approaches utilizing biological risk factors for CNS relapse, such as circulating tumor DNA, are needed to identify patients with CNS relapse.

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.