Refractory Diffuse Large B-cell Lymphoma Research Articles

CD19 Intron Retention Is Mechanism of CAR-T Treatment Resistance in Non-Hodgkin Lymphoma

Background: Chimeric antigen receptor T-cell (CAR-T) therapy has become a viable treatment option for patients (pts) with relapsed or refractory diffuse large B-cell lymphoma (R/R DLBCL). Transcriptomic markers for CD19 directed CAR-T failure have been characterized in pre-treatment B-cell acute lymphoblastic leukemia (B-ALL) but not in DLBCL. Here, we report the identification of RNA alternative splicing (AS) events associated with CD19 directed CAR-T response in pre-treatment DLBCL pts and of RNA binding proteins (RNABP) regulating these AS events. Methods: To identify putative AS markers for CAR-T treatment outcome, we performed differential AS on bulk RNA-seq samples of pre-treatment tumor biopsies in pts that had a durable response (DR), defined via remission up to 9 months post-treatment, and pts that had a non-durable response (NDR) with rMATS-turbo (Shen et al. PNAS. 2014). Intron retention (IR) events within CD19 were quantified with a secondary, more robust algorithm to ensure reproducibility and accuracy. Survival analyses were done using the survival and survminer libraries in R. Validation assays for the downstream effects CD19 IR were conducted in EJ1, Jeko-1, OCI-Ly3, SU-DHL-6, and Toledo cell lines. The surface protein expression of CD19 was assayed via flow cytometry (FC) followed by Quantibrite PE (BD Biosciences, San Jose, CA) quantification. Total CD19 protein expression was measured via western blot (WB) in the aforementioned cell lines. Analyses of CD19 IR was done via RT-PCR followed by electrophoresis, and bands were quantified with iBright Analysis Software (Thermo Fisher Scientific, Carlsbad, CA). The effect of RNABP was assessed via the creation of CRISPR/Cas9 knockout (KO) cell lines followed by WB of the target gene and quantification of CD19 surface protein expression by FC. Results: Introns 2 and 6 of CD19 are more highly expressed in NDR pts. A combined metric estimating the proportion of normal CD19 isoforms that do not express introns 2 or 6 (normal CD19) was created and DR pts expressed normal CD19 isoforms significantly higher than NDR pts (Figure 1). Normal CD19 isoforms classification performance was assessed via leave one out cross validation and achieved an overall accuracy of 67.6%. Sensitivity (classifying NDR) was 61.1% and specificity (classifying DR) was 81.3%. The expression of normal CD19 relative to the median level within the cohort was significantly associated with progress free survival (Figure 2). The expression of CD19 intron 6 was negatively correlated with CD19 surface protein expression in DLBCL cell lines. Cell lines were sorted by cells that exhibited high CD19 surface expression (bright) and low expression (dim). Cell lines sorted into the dim category expressed higher levels of intron 6 than bright cells. The correlation of RNABP expression with CD19 intron 6 expression was done in this patient cohort and the NCICCR-DLBCL cohort. Six RNABP were significantly correlated in both cohorts (r 2 > 0.2 and p-value <0.01). NOP2, one of the RNABP that was positively correlated with intron 6 expression, was knocked out in Jeko-1 cell lines to validate the putative correlation with CD19 protein expression. The resulting knock out cells exhibited lower levels of intron 6 retention and higher CD19 protein expression by FC and WB than non-transformed cells. Conclusion: Pre-treatment tumors of R/R DLBCL patients who relapse before 9 months following CD19 directed CAR-T therapy have higher expression of CD19 isoforms that retain introns 2 and 6. The upregulated expression of the CD19 isoforms retaining these introns is correlated with CD19 surface protein expression and is a marker for sustained remission following CAR-T treatment. NOP2 is correlated with CD19 intron retention and surface protein expression and could be a target for modulating CD19 expression in conjunction with CAR-T treatment. Figure 1: The expression of normal CD19 isoforms in pre-treatment tumor samples of DR (red) and NDR patients (blue). ΔPSI denotes the difference in medians between the two groups. P-value yielded by a Wilcoxon test between the two groups is reported. Figure 2: Progress free survival (PFS) stratified by expression of normal CD19 isoforms. Red - above cohort median expression of normal CD19 isoforms. Blue - below cohort median expression of normal CD19 isoforms. P-value yielded via log-rank test.

Molecular Determinants of Response to Mosunetuzumab Plus CHOP in Patients with Previously Untreated (1L) Diffuse Large B Cell Lymphoma (DLBCL)

Co-lead authors: Habib Hamidi and Patrick Kimes Background: Standard induction therapy cures ~60% of patients (pts) with DLBCL; however, most remaining non-responders die from lymphoma, highlighting a need for improved first-line treatments. Biomarkers that can reliably assess the efficacy of new treatments are lacking in clinical practice. Mosunetuzumab (Mosun) is a CD20xCD3 T cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells. In a Phase Ib/II trial (NCT03677141), Mosun combined with CHOP chemotherapy (M-CHOP) induced high response rates in pts with 1L DLBCL (ORR 87.5%, CR 85%; Phillips et al. ASH 2020). Here, we sought to identify biomarkers associated with M-CHOP response. Methods: Pts with 1L DLBCL receiving M-CHOP (six cycles) in NCT03677141 and with available biomarker data were included. Bulk RNASeq data from pre-treatment tumor biopsies were analyzed using gene set variation for tumor intrinsic pathways and xCell cell-type enrichment for immune and stromal cell types. High or low expression of each gene signature was determined by median cut-off. Cox regression examined associations between gene signatures and PFS. To assess specificity to M-CHOP, gene signatures were examined in pre-treatment biopsies from pts treated with rituximab + CHOP chemotherapy (R-CHOP) in GOYA (NCT01287741; Sehn et al. J Hematol Oncol 2020). Peripheral blood biomarkers were evaluated by whole blood flow cytometry and plasma cytokines by ELISA. Circulating tumor (ct) DNA levels were measured at baseline and on treatment using the AVENIO NHL CAPP-Seq assay (Stokowski et al. ASH 2022). Results: In M-CHOP-treated pts (n=33), gene expression analysis revealed two types of signatures associated with PFS: tumor immune microenvironment signature (TIMS) and tumor intrinsic proliferation signature (TIPS; Figure). High TIMS expression (e.g., CD4+ T cells) was associated with significantly longer PFS than low expression. Conversely, pts with high TIPS expression (e.g., MYC targets, DNA repair) had shorter PFS than those with low expression. These biological associations were specific to M-CHOP and were not observed in R-CHOP-treated pts. Consistent with the tumor-gene expression analysis, immune profiling of peripheral blood suggested that in pts treated with M-CHOP, a higher T cell baseline count was associated with a trend for prolonged PFS compared with pts with a lower baseline count. A higher count of circulating suppressive T cells was also associated with shortened PFS. Early on-treatment pharmacodynamic (PD) changes were observed following the first Mosun dose, including: induction of TNFα and IL-6, transient margination of CD4+ and CD8+ T cells, and activation of CD4+ and CD8+ T cells. The magnitude of the PD effects were similar to observations with Mosun monotherapy in pts with relapsed or refractory DLBCL (Hernandez et al. ASH 2019), confirming that Mosun maintains its immune activation effect when combined with CHOP in pts with 1L DLBCL. High baseline ctDNA levels were associated with high IPI score (n=32, Wilcoxon rank-sum p=0.007), ABC/non-GCB DLBCL subtype (n=30, p=0.033), and high lactate dehydrogenase (n=32, p=0.004). Pts with baseline ctDNA levels < median had a modest trend for improved PFS compared with pts > median (unadjusted hazard ratio [HR]=0.57 [95% CI 0.16, 1.75]; IPI-adjusted HR=0.72 [95% CI 0.16, 3.20]). The proportion of pts with undetectable ctDNA increased with each M-CHOP cycle (C) (C2 day [D]1, 25% [8/32]; C3D1, 69% [20/29]; C5D1, 88% [22/25]). Pts with undetectable ctDNA at any on-treatment time point (C2D1, C3D1, C5D1) had superior PFS compared with pts with detectable ctDNA (unadjusted HR=0.12 [95% CI 0.03, 0.52]; IPI adjusted HR=0.14 [95% CI 0.03, 0.61]). Conclusions: Consistent with the Mosun mode of action, pts with high expression of TIMS and those with high levels of circulating T cells had improved PFS versus pts with low expression. In contrast, there was no association between immune cell signatures and PFS in pts treated with R-CHOP, suggesting that the different mode of action of rituximab versus Mosun may influence this. Future studies are warranted to confirm the potential clinical utility of immune cell signatures as biomarkers associated with CD20xCD3 bispecifics' activity.

Disease Control Affects Outcomes of Patients with Relapsed Diffuse Large B Cell Lymphoma Treated with CD19 Directed CAR T-Cell Therapy

Introduction: CAR T-cell therapy against CD19 (CART-19) has become the standard of care for pts with primary refractory or relapsed diffuse large B-cell lymphoma (DLBCL) in the second and third line settings. The use of CART-19 therapy improves clinical outcomes compared to standard chemotherapy-based salvage regimens. However, DLBCL pts with disease progression following CART-19 therapy have poor overall survival (OS). Identifying biomarkers associated with improved outcomes can inform the optimal timing for CART-19 implementation. We report the impact of disease control at the time of CART-19 therapy on clinical outcomes. Methods: We conducted a retrospective study of relapsed/refractory (r/r) DLBCL treated at our institution with commercial CART-19 products between 4/2018 and 2/2023. Clinical, pathologic, and pharmacologic (type of bridging, lymphodepletion) characteristics were collected. PET/CT imaging prior to CART-19 therapy was used to determine pretreatment disease status (CR, PR or chemo-resistant disease). Treatment outcomes included best response to CART-19 therapy, frequency and severity of adverse events (CRS or ICANS), PFS and OS. Associations with clinical outcomes were evaluated using logistic regression models by Firth's method. Odds ratios [OR] and 95% CI were obtained. OS and PFS were summarized via Kaplan-Meier method; estimates of median 6- and 12-m rates were obtained with 95% CI. Associations with OS and PFS were evaluated using Cox regression models. Analyses were conducted in SAS v9.4; significance level 0.05. Results: 94 pts were included in this study. Median age was 64 yrs [range 22-81]; 66% were male; 90% Caucasian. Comorbidity index [assessed by HCT-CI] was 2+ in 51.7% pts. Median time from CART-19 discussion to re-infusion was 3.1 m [range 0.2-80 m]. Based on PET/CT scan assessment prior to CART-19 therapy, 9.6% pts achieved CR to bridging, 19% had PR, 64% were resistant (SD or PD). High risk IPI score was present in 23.4%. 14.9% pts had prior ASCT. FluCy lymphodepletion was given in 83% pts. Best response to CART-19 therapy: CR 45.7%, PR 12.8%. PD and SD observed in 28.7% and 3.2% of pts respectively. At median follow up of 36.5 m, median PFS was 6.7 m [95% CI 3.8-18.4]; median OS was 16.0 m [95% CI 9.8-30.6]. Any grade CRS occurred in 61.7%; Grade 3+ CRS 8.5%. Any grade ICANS occurred in 45.7%; Grade 3+ ICANS 16%. Second cancers occurred in 9.6%; 4 pts developed MDS. Adequate disease control at the time of CART-19 therapy was associated with improved ORR (P=0.028). Lack of disease control determined by PET/CT at the time of CART-19 infusion correlated unfavorably with OS and PFS. Pts with chemoresistant disease at the time of CART-19 therapy had a median survival of 7.2 m [HR 2.07 (0.74-5.79)] compared to pts in CR or PR at the time of CART-19 (median survival 27.9 m and 26.8 m respectively, p=0.023). Any grade ICANS [HR 2.46 (1.43-4.23) p=0.001], and ICANS grade 3+ [HR 3.38 (1.82-6.26) p=<0.001] were associated with an increased risk of death. On analysis of associations with PFS, lack of response to CART-19 therapy [SD/NR HR 15.05 (3.92-57.74) p=<0.001], any grade ICANS [HR 1.74 (1.06-2.87) p=0.029], and ICANS grade 3+ [HR 2.52 (1.38-4.59) p=0.003] were associated with increased risk for progression. Grade 3+ ICANS was adversely correlated with PFS in CR pts [HR 3.81 (1.22-11.90) p=0.021] and CR + PR pts. Several factors were associated with increased risk for toxicities. Lack of disease control at the time of CART-19 therapy, [OR 3.30 (0.79-13.79) p=0.003], development of ICANS, any grade [OR 4.08 (1.64-10.14) P=0.003] or grade 3+ ICANS [OR 7.71 (1.28-46.27) p=0.025] correlated with the development of any grade CRS. ICANS of any grade [OR 6.92 (1.12-42.79) p=0.037] and ICANS grade 3+ [OR 6.57 (1.50-28.77) p=0.013] correlated with Grade 3+ CRS. Lack of disease control status [OR 4.18 (0.85-20.55) p=0.011], any grade CRS [OR 4.08 (1.64-10.15) p=0.003], and grade 3+ CRS [OR 6.92 (1.03-46.64) p=0.047] were associated with an increased incidence and severity of ICANS. Conclusions: Disease control assessed by functional imaging at the time of CART-19 therapy for r/r DLBCL may be an independent predictor of CART-19 therapy outcomes. In this cohort, adequate disease control prior to CART-19 therapy was associated with improved ORR, PFS and OS. Optimizing disease control prior to cellular therapy may ultimately improve clinical outcomes while mitigating cellular therapy related toxicity.

Zanubrutinib Combined Regimen As Bridging Treatment on Promising Clinical Outcome of CD19 Chimeric Antigen Receptor T-Cell Therapies in Relapsed or Refractory Large B-Cell Lymphoma

Background Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) shows poor clinical outcomes. Anti-CD19 chimeric antigen receptor-T cells (CAR-T) therapy is emerging as the second-line therapy of choice, but in real-world studies its efficacy and long term follow is not promising. Effective bridging therapy can reduce tumor burden and improve CAR-T efficacy. Zanubrutinib, a selective Bruton tyrosine kinase (BTK) inhibitors, combined regimens have established therapeutic activity by targeting B-cell receptor signaling and have individually shown efficacy in patients with R/R DLBCL. Thus, the present study evaluated the efficacy and safety of zanubrutinib-combined regimen as bridging treatment in CD19 CAR-T therapy. Methods This retrospective study includes all patients participant in the clinical trail (NCT 02537977) between January 2020 and December 2022. All 21 heavily treated patients with r/r DLBCL who received zanubrutinib-combined regimens as bridging therapy are included. Patient clinical characteristics were collected upon patient enrollment. Efficacy outcomes included overall response rate (ORR), progression-free survival (PFS), and overall survival (OS), whereas safety outcomes included incidence of adverse events. The zanubrutinib combined regimens are zanubrutinib with IMIDs, chemotherapy and/or rituximab. Results: Of the 21 evaluable patients, median age of patients is 57 year old and median treatment line is 3. 19 patients (90%) are non-GCB subtype and 18 patients(86%) are IPI score >3. 14 patients(67%) are double expression and 7 patients (33%) have TP53 mutation. Objective response rate(ORR) is 81%; of whom, 10 patients(48%) got complete remission(CR) and 7 patients(33%) got partial remission(PR). With a median follow-up of 25 months, the median PFS was 12.8 months and median OS was not yet reached. 8 CR patients and 3 PR patients maintain long-term remission and are still under followup. 18 patients (86%) has grade 1-2 cytokines release syndrome(CRS) and 3 patients(14%) has grade 1 immune effector cell-associated neurotoxicity syndrome (ICANS). No severe CRS and >1 grade ICANS were observed. The most common hematologic toxicities included grade ≥3 neutropenia (90%) and grade 3/4 thrombocytopenia (71%). No patients had grade ≥3 non hematologic toxicities. Conclusion: Zanubrutinib-combined regimens are effective and safe as bridging therapy in CD19 CAR-T therapy. More prospective studies are further command.

BTK Inhibitors and Chidamide-Combined Therapy in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background: Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) shows poor clinical outcomes, particularly in patients ineligible for stem cell transplantation or who failed induction therapy or salvage therapy. Monotherapy Bruton tyrosine kinase inhibitors (BTKi) have established modest antitumor effect in R/R DLBCL by targeting B-cell receptor signaling, with median progression-free survival (PFS) and overall survival (OS) of 1.64-2.8 months and 6.4-8.4 months, respectively. Chidamide is a selective oral histone deacetylase inhibitors (HDACi) that shows promising treatment for DLBCL and may overcome BTKi resistance. So, we evaluated the efficacy and safety of BTKis and chidamide-combined therapy in R/R DLBCL. Methods: This retrospective study consists of 11 R/R DLBCL who received combination therapy of chidamide and BTK inhibitors between September 2021 and July 2023. Chidamide was orally administered at a dose of 20 mg twice a week, while the BTK inhibitor was administered daily. Patient clinical characteristics were collected upon patient enrollment. Efficacy outcomes included overall response rate (ORR), PFS and OS, whereas safety outcomes included incidence of adverse events. Results: 11 R/R DLBCL patients were enrolled. Median age was 61 years (range, 56-73), and 81.82% of the patients had non-germinal center B-cell-like (non-GCB) DLBCL, one had GCB DLBCL, one had transformed DLBCL. The median number of previous treatment lines was 3 (range, 3-8). 10 patients (90.91%) received treatment with BTK inhibitors before. Four patients（36.36%）failed to chimeric antigen receptor T-cell/NK-cell(CAR-T/CAR-NK) therapy. The best ORR was 81.82%, with 9 patients acquired objective disease remission after 1-2 cycles. Among them, 3 patients (27.27%) achieved complete response, and6 patients (54.55%) achieved partial remission. With a median follow-up of 5 months, the median PFS was 6.0 months (95%CI 3.14-8.86) and median OS was not yet reached. In terms of adverse events, the most common hematologic toxicities observed were grade ≥3 neutropenia (36.36%, 4/11) and grade ≥3 thrombocytopenia (36.36%, 4/11). Two patients (18.18%) had pneumonia, and one patient (9.09%) had herpes zosters. Due to the positive real-world outcomes observed in the retrospective study, prospective clinical studies of chidamide in combination with BTK inhibitors for the treatment of R/R DLBCL are underway (ChiCTR2300073233). Conclusion: This real-world study revealed promising results. Despite the majority of patients had been treated with BTK inhibitors and experienced relapse and resistance, the addition of chidamide brought patients back into remission, suggesting that chidamide can enhance the efficacy of BTK inhibitors and overcome acquired resistance. Notably, chidamide combined with BTK inhibitors showed high efficacy and manageable tolerability, making it a promising treatment option for patients with R/R DLBCL. Keywords: BTK inhibitors, Chidamide, R/R DLBCL, efficacy and safety

Real-World Use of Tafasitamab (tafa) for Relapsed or Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL) Among Racial and Ethnic Minorities in the United States

Introduction: Tafa, a CD19-targeting immunotherapy, received accelerated approval by the FDA in combination with lenalidomide (len) for the treatment of patients with R/R DLBCL ineligible for autologous stem cell transplantation. In a recent real-world study (RWS) of tafa for R/R DLBCL, nearly one-third of patients were from typically underrepresented racial groups (abstract submitted to ASH 2023). Research has shown that patient characteristics, risk factors, and outcomes for DLBCL can differ by race and ethnicity (Li Y, et al. Cancer Epidemiol. 2015;39:8-13; Ermann D et al. J Clin Oncol. 2022;40(suppl):7507). The purpose of this analysis is to understand the patient characteristics, treatment patterns, and outcomes observed in the tafa RWS by race and ethnicity. Methods: This was a retrospective cohort study in which participating physicians from Cardinal Health's Oncology Provider Extended Network abstracted data from eligible patients' medical records into electronic case report forms. Eligible patients were ≥18 years of age who initiated tafa for R/R DLBCL on or after Oct 21, 2020. Included patients had ≥4 months of follow-up unless the patient died during this period. Use of tafa in combination with len was not a requirement for study eligibility. Patients who received tafa as part of a clinical trial were excluded. Results were summarized by race (White, Black/African American, “Other”) and ethnicity (Non-Hispanic, Hispanic) using descriptive statistics. The “Other” racial category included patients with mixed race or a race other than White, Black/African American, or Unknown. The Kaplan-Meier method was used to measure time to tafa discontinuation. Due to limited follow-up and small sample size, effectiveness outcomes for subgroups smaller than 20 patients are not presented. Results: A total of 181 patients who initiated tafa for R/R DLBCL were included in this analysis. Table 1 shows patient characteristics, treatment patterns, and outcomes by racial and ethnic groups. The racial composition of the study population was 64% White, 22% Black/African American, 8% “Other,” and 6% Unknown (Unknown not displayed in Table), and ethnic composition was 82% non-Hispanic, 17% Hispanic, and 1% Unknown (Unknown not displayed in Table). Most physicians who participated in this study were from community oncology practices (83%). The median follow-up time from tafa initiation to data collection among all patients was 6.5 (interquartile range [IQR], 5.0-8.6) months. The majority of patients (96%) received tafa in combination with len. At the time of data collection, 144 patients (80%) were alive, among whom 121 (84%) were still receiving tafa. The median age at which tafa was initiated ranged from 70 to 72 years for all racial and ethnic groups. Among the racial groups, the White cohort had the highest percentage of patients with an ECOG PS ≥2 (53%) and Ann Arbor Stage IV (68%) at time of tafa initiation. Among the racial categories, the median time (IQR) from initial DLBCL diagnosis to initiation of tafa was longest in Black/African Americans (23.3 [12.6-34.8] months) and shortest in the “Other” group (18.0 [15.0-21.4] months). In non-Hispanics and Hispanics, the median (IQR) duration from initial DLBCL diagnosis to tafa initiation was 20.0 (12.4-35.0) and 19.0 (14.0-34.0) months, respectively ( Table 1). Among patients who had disease response data, the real-world overall response rate (95% CI) was 75.0% (66.8-83.2) and 77.5% (64.6-90.4) for the White and Black/African American cohorts, and 74.5% (67.3-81.7) and 80.8% (65.7-95.9) for non-Hispanic and Hispanic groups, respectively ( Figure 1). Conclusions: The diverse population in this RWS is reflective of the heterogeneous patient population with R/R DLBCL treated primarily at community oncology centers in the United States. In addition, the study results support the clinical benefit of tafa across racial and ethnic groups. The majority of patients were still receiving tafa at data cutoff and follow-up was limited in duration; the median follow-up time in this study was shorter than the median time to complete response observed in L-MIND (NCT02399085; Duell J, et al. Haematologica. 2021;106:2417-2426), the pivotal phase 2 trial that contributed to the accelerated FDA approval of tafa for R/R DLBCL. A longer follow-up is required to better assess long-term outcomes of tafa treatment for R/R DLBCL among racial and ethnic patient groups.

Improved Survival of R/R Double Hit/Triple Hit Lymphoma in the Era of CD19 Chimeric Antigen T Cell (CART) Therapy

Introduction: DH/THL also classified as high-grade B cell lymphomas (HGBCL) with MYC and BCL2 and/or BCL6 rearrangements have higher risk of progression after initial treatment and poor response to salvage therapies compared to diffuse large B cell lymphoma (DLBCL), NOS. Recently, CART cell therapy has shown encouraging responses in treatment of relapsed refractory (R/R) DLBCL. In this retrospective study, we evaluated whether survival of (R/R) DH/THL is improved in the CART era. Methods: In this multicenter retrospective study, adult pts (pts) with R/R DH/THL after anthracycline-based therapy were included. Demographic and clinical variables were obtained from electronic health records. Study objectives was overall survival (OS) at 2 year landmark. We utilized full propensity score matching to compare progression free survival (PFS) and OS of CART with 2 nd line chemoimmunotherapy. Results: 278 DH/THL pts treated between 2010-2020 were included. Median age was 63 years (range 52-74), 163 (58%) were male, 119 (43%) were classified as DLBCL and 116 (42%) as HGBCL; 40 (14%) were transformed from follicular lymphoma. 225 (82%) had advanced stage, 161 (67%) had elevated LDH, 85 (33%) had bulky disease, 74 (59%) had B symptoms. 105 (38%) pts treated for relapsed disease before 2018 were included in the pre-CART era cohort. 168 (60%) pts treated from 2018 onwards were included in the CART era cohort. 102 (60%) pts in CART cell era received CART cells, 40 (14%) received polatuzumab vedotin (pola), 10 (3.5%) received loncastuximab tesirine, tafasitamab and bispecific antibody each. Characteristics at relapse were similar in both groups. Median follow up was 8.9 (0.2-146) months. 2-year OS estimate was 36% (CI 95: 30-42%) in entire population. Patients treated in CART cell era had significantly higher OS (2 yr OS: 43% [CI 95: 35-53]) compared to pts treated in pre-CART cells era (2-year OS: 24% [CI 95: 16-34], p=0.0006) (Figure 1). In multivariable analyses, increasing age (HR = 1.02 (CI 95: 1.01, 1.04), p=0.003), extranodal disease (HR = 1.41 (CI 95: 1.00, 1.99), p<0.001), elevated LDH (HR = 1.57 (CI 95: 1.11, 2.24), p=0.01) were associated with inferior OS. Increasing time to relapse (HR = 0.98 (CI 95: 0.97, 0.99), p<0.001) was associated with superior OS. BCL6 rearrangement did not affect OS. When adjusted for these factors, treatment with CART cells was associated with higher 2 yr OS (HR = 0.33 (CI 95: 0.22, 0.49), p<0.001). Treatment with polatuzumab (HR = 1.07 (CI 95: 0.71, 1.6), p=0.07) was not significantly associated with OS in univariable analysis. 102 pts underwent CART cell therapy at any point for relapse; 94 (93%) were refractory to prior chemotherapy. 62 (60%) responded and 52 (50%) had CR after CART cells. 2-year PFS and OS after CART cell therapy were 28% (CI 95: 19-40) and 40% (CI 95: 30-53), respectively. Increasing time to CART was associated with inferior OS (HR: 1.02 (CI 95: 1.0-1.03, p=0.03) but not PFS (HR: 1.02 (CI 95: 0.99-1.04), p=0.2). 219 (79%) pts received curative intent salvage chemotherapy for 2 nd line. 2-year PFS and OS were 15% (CI 95: 11-20) and 38% (CI 95: 31-45), respectively. 21 pts received CART cell therapy for 2 nd line. 2 year PFS and OS in this small number of pts were 52% (CI 95: 35-79) and 70% (CI 95: 52-94), respectively. Due in part to the small number of pts who received CART cells in 2 nd line, we utilized full propensity score matched analysis to compare each patient in CART cell group to all possible matched controls in salvage chemotherapy group. Variables matched were age, LDH, extra nodal disease and time to relapse. In propensity score matched analysis, pts who received CART cells had significantly higher 2 year PFS (HR: 10.5 (CI 95: 3.4-17.5), p=002) and 2 year OS (HR: 7.5 (CI 95: 1.4-13), p=0.01). Conclusions: Survival of r/r DH/THL appears to have improved in the era of CART cell therapy. About 2/3 of pts refractory to chemotherapy respond to CART cells, and in multivariable analysis, receipt of CART cells was associated with improved OS. Increasing time to CART cells was associated with inferior survival. In a propensity score matched analysis utilizing full matching, CART cell therapy in 2 nd line was associated with significantly higher 2 year PFS and OS compared to curative intent salvage therapy. Other CD 19 and CD 20 targeting agents approved after CART cell therapy might contribute to favorable survival, effect of these agents could not be assessed because of small numbers.

Co-Expression of C-MYC/BCL2 Is Associated with Inferior Survival Outcomes in Relapsed/Refractory Diffuse Large B-Cell Lymphoma after Autologous Stem Cell Transplantation - a Nationwide Retrospective Analysis in Singapore

Introduction Co-expression of C-MYC and BCL2 in diffuse large b-cell lymphomas (DLBCL), also termed double-expressor lymphomas (DEL), has been shown to be associated with poorer outcomes after standard R-CHOP induction therapy. Approximately 70% of DEL will relapse within 5 years. The subsequent traditional approach with potential for cure has been high dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT). Whilst factors such as remission status after front-line treatment and early relapse have been associated with poorer outcomes after ASCT, data are limited regarding significance of DEL status on survival outcomes post-ASCT. We retrospectively studied the prognostic impact of DEL status on outcomes in patients with relapsed or refractory (R/R) DLBCL who underwent ASCT. Methods This was a retrospective, nationwide study of adults patients, >18 years old, with R/R DLBCL who underwent autologous hematopoietic stem cell transplantation (ASCT) between 2010 and 2022 at the only 2 transplant centers in Singapore - Singapore General Hospital and National University Cancer Institute. Patients with double/triple hit lymphoma, defined as concurrent rearrangements of MYC and BCL2 and/or BCL6 by fluorescence in situ hybridization (FISH) analysis, were not included in this study. Patients with transformed indolent B-cell NHL (non hodgkin lymphoma), primary mediastinal B-cell lymphoma, primary CNS (central nervous system) lymphoma, or Richter transformation of chronic lymphocytic leukemia were excluded. Only patients that had available MYC and BCL-2 immunohistochemistry (IHC) were included in this study. Overall survival (OS) and progression free survival (PFS) were estimated using the Kaplan-Meier method. Cox proportional hazards regression models were used to assess associations between covariates of interest and OS or PFS. Results A total of 72 patients were included, 44 (61.1%) non-double expressor DLBCL (non-DEL) and 28 (38.9%) double expressor DLBCL (DEL). Patient demographics and disease characteristics at relapse were similar in both groups aside from a higher incidence of CNS involvement in the non-DEL group as compared to DEL group, 34.1% vs 10.7% (Table 1). Both groups achieved a >90% response (CR and PR) after second-line salvage chemotherapy as well as a >90% response post-transplant (CR and PR). Patients with DEL however were more likely to relapse post-transplant, 69.7% vs 40.9% (p = 0.032) and were also associated with increased odds of all-cause mortality, 60.7% vs 36.4% (p=0.043). This significance was maintained on multivariable analysis, HR 4.22 95% CI 1.20 - 14.84 (p=0.025) and HR 4.32 95% CI 1.10 - 17.15 (p=0.038) respectively. The median follow up for survivors were 42 months (range, 26 to 64 months) in the DEL patients and 60 months (range, 46 to 88 months) in the non-DEL patients. The 5-year PFS in patients with DEL was inferior to non-DEL patients, 23.3% vs 49.6% respectively, p=0.022. DEL patients were also associated with inferior 5-year OS, 40.2% vs 65.7%, p=0.041. In multivariable model, DEL status remained significantly associated with inferior PFS and OS. We also observed that patients with CNS involvement at relapse had inferior PFS and OS. Male gender and a less than complete metabolic response (CR) status prior to transplant, assessed on PET-CT, was also associated with inferior OS. Conclusion Co-expression of C-MYC/BCL2 was associated with inferior outcomes after ASCT in patients with R/R DLBCL. While ASCT has been the standard curative approach in the R/R setting, considerations for novel targeting therapies to achieve a deeper response prior to transplant or the upfront use of chimeric antigen receptor T-cell therapy at first relapse may be further explored in this high-risk subset of patients.

Optimizing Circulating Tumor DNA Limits of Detection for DLBCL during First Line Therapy

Background: Circulating tumor DNA (ctDNA) assessment is effective in diffuse large B-cell lymphoma (DLBCL) monitoring and risk stratification, with prognostic utility throughout first-line (1L) therapy. DLBCL ctDNA assays vary in analytical sensitivity, or limit of detection (LOD), which range from parts per thousand to below 1 part per million (1 in 10 6). With differing assays and time-points assessed in DLBCL studies, the relationship between analytical and clinical sensitivity for outcome prediction remains unclear. We assessed the prognostic ability of ctDNA at various LODs and used modeling strategies to project the efficacy of assays for minimal residual disease (MRD) detection. Methods: We previously reported a DLBCL dataset assessing plasma ctDNA before, during and after curative-intent 1L anthracycline-based treatment from multiple prospective studies, including RCHOP or EP[O]CH with acalabrutinib, lenalidomide, obinutuzumab, and polatuzumab (Roschewski et al. ASH 2022). In this study, ctDNA was evaluated by Phased Variant Enrichment Detection and Sequencing (PhasED-Seq, Foresight Diagnostics), a ctDNA MRD assay with LOD below 1 in 10 6. Samples from this study and a prospective DLBCL cohort treated with standard 1L therapy at Samsung Medical Center were considered in this analysis. We assessed the prognostic ability of ctDNA detection, considering LODs from 1 in 100 (10 2)to 1 in 10 6 to predict progression free survival (PFS) before, during and after 1L treatment. We assessed the desired sensitivity for ctDNA MRD during treatment by generating patient-specific log-linear models assuming exponential decay. We projected the distribution of variant allele frequencies (VAFs) during therapy from cycles 2 to 5 for patients who progressed to determine the minimal acceptable analytical sensitivity. Results: We included 230 patients consisting of 588 ctDNA samples, with 201 before therapy, 71 at C2D1, 101 at C3D1, 70 at C4D1 and 145 at end of therapy (EOT). Median follow-up was 17.5 months and 62 patients (27%) progressed. To evaluate the impact of LOD at treatment milestones, we applied a threshold for ctDNA positivity ranging from 1 in 10 2 to 1 in 10 6. Increased LOD had no effect on the performance of ctDNA before therapy (Figure A & B). At C2D1, if the LOD was at least 1 in 10 4, there was no difference in MRD prognostic performance. Starting at C3D1, improving the LOD for ctDNA positivity down to 1 in 10 6 showed superior predictive power for PFS at 24 months. The power of ctDNA to predict PFS at 24 months improved later in therapy, with area under the receiver operator curves (AUROCs) for PFS24 of 0.68, 0.73, 0.77, 0.88, and 0.86, at pretreatment, C2D1, C3D1, C4D1, and EOT time-points respectively (Figure A). To further understand the desired LOD for ctDNA MRD, we developed personalized models of ctDNA VAFs. Exponential models fit the data well in 43/44 cases with ≥ 3 MRD-detectable samples through C4D1, with a median correlation of 0.91, confirming their utility for modeling ctDNA. We generated log-linear models for 106 patients with ≥ 2 MRD-detectable samples, and used log-fold change in VAF per cycle (i.e. the slope) to compare patients by progression. Median log-fold change in VAF per cycle was worse for patients who progressed at -1.1 (IQR -1.4, -0.7) compared to those who remained disease-free with median -1.5 (IQR -2.0, -1.1) (p<0.0001). Patients with primary refractory DLBCL on EOT imaging had less robust log-fold change per cycle with median of -0.9 (IQR -1.3, -0.5) compared to those who relapsed after CR with median -1.3 (IQR -1.7, -0.9) (p=0.0004). We used the distribution of slopes to project VAFs for cycles 2 to 5 for patients who progressed to determine the LODs that provide acceptable clinical sensitivity. We found the analytical sensitivity for detecting DLBCL extended lower for each cycle, demonstrating the need for a LOD at least 1 in 10 6 for robust MRD detection at late time-points during 1L treatment. Conclusion: When using an ultrasensitive assay, MRD assessment at later timepoints better predicts PFS than at early timepoints. While the technical LOD does not affect disease burden assessment before therapy, using more sensitive assays during and after therapy improves disease detection and outcome prediction. Utilizing the most sensitive ctDNA MRD assays in 1L DLBCL therapy will maximize the efficacy of MRD-driven therapeutic strategies and MRD as a surrogate endpoint in future trials.

CAR T-Cell Therapy Combined with PD-1 Inhibitors Significantly Improve the Efficacy and Prognosis of r/r DLBCL with TP53 Alterations

More than 40% of patients diagnosed with diffuse large B-cell lymphoma (DLBCL) eventually develop relapsed or refractory (r/r) disease and typically have poor outcomes. Autologous chimeric antigen receptor T-cell therapy (CART) has emerged as a crucial second line treatment option for r/r DLBCL, but 60-70% of patients still relapse or are refractory. Recent research has shed light on the role of TP53 alterations (mutation or deletion) in r/r DLBCL patients who do not respond well to CART cell therapy. The findings in this study will show that the combination of CART with PD-1 inhibitors (ICIs) had a significant impact on the efficacy and prognosis of r/r DLBCL patients with TP53 alterations. This retrospective analysis included adult DLBCL patients who were treated at Shanghai Tongji Hospital of Tongji University with autologous CD19 (N=20) or CD20 (N=2, patient 6 and 7, Fig.1 A) CART-cell therapy between January 1, 2019, and September 30, 2022. All enrolled patients provided their informed consent to participate in the study. The protocol of the study was reviewed and approved by the Institutional Review Board of the National Cancer Institute, ensuring ethical practices were followed. A total of 22 patients with TP53 alterations undergoing CD19-CART therapy were included, of which 2 patients received CD20 CART cell after recurrence of CD19 CART cell therapy. The total dose of CD19 or CD20-CAR T-cell is between 1- 3x10^6/kg. Sintilimab/Tislelizumab was used as PD-1 inhibitors (3 mg/kg in patients weighing <60 kg or 200 mg in patients weighing ≥60 kg) every three weeks within 6 weeks after CAR-T infusion. 1 case had grade 3-4 Cytokine Release Syndrome (CRS) and no case had CART-cell-related encephalopathy syndrome (CRES). In Table 1, the patients are divided into two groups. One group received CART cells combined with PD-1 inhibitors (ICI+), while the other group received CART cells only (ICI-). The median ICIs treatment time after CART infusion was 17 days (IQR 11-38 days). Baseline factors such as age, gender, disease severity, previous treatment history and etc. had no substantial differences between the groups. Treatment regimen and the subsequent response were shown in Fig.1 A and B. Patient 1-9 received ICIs treatment after CART cell therapy, of which 8 cases (88.9%) showed the best objective response rate (ORR) at day 90 after CART infusion (Fig.1B) and 6 cases (66.7%) achieved complete remission (CR). In comparison, among the 13 individuals who were treated with CART alone, only 3 cases (23.1%) showed the best ORR at day 90 after CART infusion and only 1 case (7.7%) achieved complete remission. We found a significant difference between the two treatment groups by performing a Chi-square test (P=0.009). Our results suggest that the addition of PD-1 inhibitors to the CART regimen significantly improves the treatment outcome in patients with TP53 alterations. In Fig.1 C and D, we show that the median progression-free survival (PFS) was NR (not reached) vs. 1.7 months (95% CI:1.0 month-2.3 months) and the median overall survival (OS) was NR vs.10.9 months (95% CI: 3.8 months-18.1 months) in the ICI+ and the ICI- groups. The p-values for PFS and OS was 0.001 (HR=0.18, 95% CI: 0.07-0.47) and 0.003 (HR=0.15, 95% CI: 0.05-0.42) between the two groups. These results suggest that patients in the combination treatment group experienced less disease progression and were more likely to survive longer. Overall, our study demonstrates the potential benefits of combining immunotherapies, specifically CART with PD-1 inhibitors, in the treatment of relapsed or refractory DLBCL patients with TP53 alterations.

Factors for predicting treatment success and severe adverse events of chimeric antigen receptor (CAR) T-cell therapy in relapsed or refractory diffuse large B-cell lymphoma

Factors for predicting treatment success and severe adverse events of chimeric antigen receptor (CAR) T-cell therapy in relapsed or refractory diffuse large B-cell lymphoma

CEAC (oral semustine, etoposide, cytarabine and cyclophosphamide) vs BEAM (carmustine, etoposide, cytarabine, and melphalan) conditioning regimen of autologous stem cell transplantation for diffuse large B-cell lymphoma: a post-hoc, propensity score-matched, cohort study in Chinese patients.

Autologous stem cell transplantation (ASCT) is a salvage therapy for relapsed or refractory diffuse large B-cell lymphoma (DLBCL). We have developed a novel conditioning regimen called CEAC (oral semustine 250mg/m2 d-6, etoposide 300mg/m2 d-5 ~ d-2, cytarabine 500mg/m2 d-5 ~ d-2, and cyclophosphamide 1200mg/m2 d-5 ~ d-2) In lymphoma patients in China. Here, we conducted a study to compare the conventional BEAM regimen with the CEAC regimen in 110 DLBCL patients. Propensity-score matching was performed in a 1:4 ratio (22 patients received BEAM and 88 received CEAC). Our results showed no significant difference in the overall response rate (95% vs 97%, P = 1.000) and complete response rate (66% vs 73%, P = 0.580) between the two cohorts. The 5-year progression-free survival (PFS), 5-year overall survival (OS), and 5-year cumulative incidence of relapse (CIR) for all patients were 72% (95% CI 62%-82%), 92% (95% CI 86%-97%), and 29% (95% CI 17%-38%), respectively. There was no significant difference in the 5-year PFS (80% vs 70%, P = 0.637), 5-year OS (95% vs 91%, P = 0.496), and 5-year CIR (20% vs 30%, P = 0.733) between cohorts. In terms of safety, the CEAC cohort had a lower incidence rate of grade 1-2 gastrointestinal hemorrhage (P = 0.023) and severe nausea (P = 0.007) compared with the BEAM cohort. In conclusion, the CEAC regimen seems to be a suitable alternative to the BEAM regimen for ASCT in DLBCL patients.

A Phase I Study of CD19-Targeted 19(T2)28z1xx CAR T Cells in Adult Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background: Autologous CD19 CAR T cell therapies have demonstrated favorable clinical responses in relapsed or refractory (R/R) diffuse large B-cell lymphomas (DLBCL), but only a subset of patients (pts) experience durable remissions. We hypothesized that the redundancy of CD28 and CD3ζ signaling in a CAR design incorporating all 3 CD3ζ immunoreceptor tyrosine-based activation motifs (ITAMs) might negatively affect T cell differentiation and promote exhaustion. Therefore, we created a new CD19 CAR construct with calibrated CAR activation potential by mutating 2 of the 3 ITAMs, termed 1XX, and hypothesized that a 1XX CAR would afford durable responses at lower dose and fewer toxicities compared to CARs comprising 3 ITAMs. We previously presented the data from the dose-escalation portion of the phase I clinical trial of 19(T2)28z-1XX CAR T cells in DLBCL (Park J et al. ASH 2022). We have now completed accrual and report the updated results from both dose escalation and expansion portion of the study with a longer follow-up (NCT04464200). Methods: This is a single-center, phase I, dose-escalation and expansion trial of 19(T2)28z1XX CAR T cells in adult pts with R/R DLBCL conducted at Memorial Sloan Kettering Cancer Center. Pts received fludarabine and cyclophosphamide lymphodepleting chemotherapy followed by a single infusion of CAR T cells. There were 4 dose levels in the dose escalation cohort: dose level (DL) 1 (25x10 6), DL2 (50x10 6), DL3 (100x10 6) and DL4 (200x10 6 CAR T cells). The primary objective of the trial was to evaluate safety and tolerability and determine the recommended phase 2 dose. Key secondary objectives include assessment of overall response and complete response rates. Results: 28 pts have been enrolled and treated; 16 pts on the dose escalation and 12 pts on the dose expansion phase of the study. Median age was 62 (range, 48-86). 20 pts (71%) had primary refractory disease, 3 pts (11%) had prior autologous HSCT, and 2 pts (7%) had prior autologous CD19 CAR T therapy. In the dose-escalation phase (n=16), 15 pts experienced grade 1-2 CRS (94%) and no pt experienced ≥grade 3 CRS. Two pts (13%) experienced ICANS: 1 pt with grade 1 and 1 pt with grade 3 (Table 1). Eight pts received tocilizumab and 5 pts received corticosteroid. Complete response (CR) was observed in 11 of 16 pts (69%) and partial remission (PR) in 1 pt (6%) with overall response rate (ORR) of 75%. Responses were seen across all dose levels (Table 1). The lowest dose (DL1: 25x10 6) was selected for dose-expansion based on exposure-response analysis and 12 additional pts were treated at that dose level. In a pooled analysis of 16 pts who received DL1 dose (4 in dose escalation and 12 in dose expansion), 12 pts and 1 pt experienced grade 1-2 (75%) and grade 3 CRS (6%), respectively. Only 1 pt experienced ICANS (6%) at grade 3 (same pt who had grade 3 CRS), which was reversible and transient (Table 1). 11 pts received tocilizumab and 9 pts received corticosteroids. Twelve pts achieved CR (75%) and 2 pts (12%) PR with the best ORR of 87% (Table 1). A lower metabolic tumor volume (MTV) at screening but not at post-bridging was associated with a higher likelihood of achieving CR (p=0.039). With a median follow-up of 345 days (range, 33-1024), 4 pts experienced relapse (1 CR and 3 PR pts) and 16 pts have ongoing CR > 6 months (Figure 1). While CAR T cell persistence analysis is ongoing, 5 pts have CAR T cells detected beyond one year in ongoing CR including a patient treated at DL1 with detectable CAR T cells at 2 years. Conclusions: Treatment with 19(T2)28z1XX CAR T cells was shown to be safe with durable remissions with low rates of severe CRS (4%) and ICANS (7%). The overall CR rate of 71% is encouraging and appears durable with the majority of CR patients in ongoing remission > 6 months at a median follow-up of 345 days and with persisting CAR T cells. With more patients treated at DL1 with a longer follow-up, the study demonstrates a high efficacy of 19(T2)28z1XX CAR T cells at a dose significantly lower (25x10 6) than those of approved CD19 CAR T cells (100-300x10 6) and support that the 1XX single ITAM signaling domain may lead to enhanced efficacy of CD19 CARs and allow infusion of a lower T cell dose with favorable toxicity profiles.

Loncastuximab Tesirine Demonstrated Substantial Single-Agent Efficacy and Manageable Safety Profile in Heavily Pretreated Chinese Patients with Relapsed or Refractory (R/R) Diffuse Large B-Cell Lymphoma (DLBCL)

Introduction: Patients with R/R DLBCL who failed multi-agent chemoimmunotherapies usually have a poor prognosis, leaving a significant unmet medical need. Loncastuximab tesirine (Lonca) is an antibody-drug conjugate, composed of a humanized anti-CD19 antibody conjugated to a pyrrolobenzodiazepine dimer toxin that showed clinical efficacy and manageable safety in heavily pretreated DLBCL patients in a global phase 2 study of Lonca monotherapy (NCT03075696). Here we present results from a phase 2 study (ChiCTR2300072058) of Lonca monotherapy in Chinese patients with R/R DLBCL for the first time, including analyses of responses in high-risk DLBCL subgroups. Methods: This open-label, single-arm, phase 2 study was designed to confirm the safety, efficacy and PK profiles of Lonca monotherapy in Chinese patients with R/R DLBCL who had failed ≥ 2 lines of systemic therapies. The primary endpoint was overall response rate (ORR) as assessed by IRC according to the Lugano 2014 criteria. Secondary efficacy endpoints include complete response rate (CRR), DOR, RFS, PFS, and OS. Treatment-emergent adverse events (TEAEs) were reported by CTCAE v4.0. Results: As of data cutoff (11-Jan-2023), 64 Chinese patients with DLBCL were enrolled and received at least one dose of Lonca (median: 4.0 cycles [range: 1-17]) and were evaluable for safety and efficacy analyses. The median number of prior lines of therapies was 3.0 (range: 2-12), with 67.2% of patients having ≥ 3 lines of prior therapies. Four patients (6.3%) had received prior CD19 CAR-T therapy. Forty-three patients (67.2%) were refractory to the first-line therapy (primary refractory), 56 patients (87.5%) were refractory to the most recent line of therapy and 40 patients (62.5%) were refractory to all prior lines of therapies. At data cutoff, the median follow-up was 8.5 months. The ORR by IRC was 51.6% (95% CI: 38.7% to 64.2%), and CRR was 23.4%. The median DOR (mDOR) was 6.37 months as assessed either by IRC or by investigator. For patients with a CR, the mDOR was 10.22 months (6.08 months for patients with PR). The median time to first response was 41.0 days. The median PFS and median OS were 4.96 and 9.33 months, respectively. Responses, including CRs, were observed in several high-risk subgroups (Fig. 1): ORR was 54.7% in patients with advanced stage disease (Stage III/IV) and 50.0% in patients with transformed disease. Patients who were refractory to first-line therapy, to the most recent therapy, or to all prior therapies had ORRs of 44.2%, 51.8% and 42.5%, respectively. Three patients who had received a prior SCT had an ORR of 100%. Lonca was also effective in elderly patients ≥ 65 years (ORR of 45.8%, mDOR of 6.37 months) and in patients who had received prior CD19 CAR-T therapy (ORR of 50%). Overall, 64 (100%) patients had at least one TEAE and 61 (95.3%) patients had at least one TEAE ≥ Grade 3. The most common (≥30%) all-grade TEAEs were GGT increased (71.9%), followed by anaemia (70.3%), platelet count decreased (65.6%), AST increased (64.1%), WBC count decreased (64.1%), neutrophil count decreased (60.9%), ALT increased (51.6%), hypokalaemia (37.5%), and blood ALP increased (32.8%). The most common (≥15%) TEAEs of Grade ≥3 were platelet count decreased (34.4%), neutrophil count decreased (28.1%), WBC count decreased (28.1%), GGT increased (25.0%), anaemia (18.8%), hypokalaemia (18.8%), neutropenia (17.2%), and lymphocyte count decreased (15.6%). The rate of febrile neutropenia was low (3.1%). The majority of Grade ≥ 3 TEAEs were abnormalities of clinical laboratory values as opposed to clinical symptoms and were generally reversible. Treatment-related TEAEs leading to treatment discontinuation occurred in 10 patients (15.6%), most commonly GGT increased (3 patients; 4.7%). No increase in toxicity was seen in patients aged ≥65 years compared with younger patients. Conclusions: Lonca demonstrated substantial and clinically meaningful single-agent efficacy and was well-tolerated in heavily pretreated Chinese patients with R/R DLBCL. Toxicities were generally manageable and reversible in most patients by routine clinical practice and/or with dose modifications, with neither unexpected safety concerns nor different toxicity profile in patients aged ≥65 years. Encouraging and durable responses were also observed in high-risk patient groups, including patients with advanced stage, transformed or refractory DLBCL.

Molecular Mechanisms Underlying Response and Resistance to Glofitamab

The prognosis for patients with relapsed or refractory Diffuse Large B-cell Lymphoma (r/rDLBCL) is poor. Glofitamab (a CD20xCD3 T-cell-engaging bispecific antibody) demonstrated high and durable complete response rates and a manageable safety profile in patients with relapsed/refractory (R/R) Large B-cell lymphoma (LBCL). Despite its clinical efficacy and a rapid achievement of complete responses, some patients are refractory to glofitamab treatment, prompting us to investigate the mechanisms underlying response and resistance to glofitamab. To gain insight into these mechanisms, we performed molecular (single cell RNA and TCR sequencing data) and functional (ex-vivo assessment of cytotoxicity, cytokine and cytotoxic granules secretion, activation) characterisation of PBMCs from glofitamab-treated patients who achieved complete response (CR, n=18) or progressed (progressive disease, PD, n=13) by cycle 3, collected in a Phase I/II study (NCT03075696). All patients received glofitamab in a step-up-dose regimen of 2.5/10/30 mg following obinutuzumab pre-treatment (1000 mg) administered 7 days prior to the first glofitamab dose. The PBMC samples were analyzed at baseline (cycle 1 day 1 pre-dose) and on treatment (cycle 2 day 1 pre-dose). The assessment of PBMCs using single cell RNA sequencing did not reveal major differences in abundance of the main immune cell subsets between patients who achieved CR or PD. Paired analysis of on-treatment samples (compared to respective baseline) pointed to a tendency towards decreased frequency of CD8 T cells in patients with CRs and increased frequency of the same in patients with PDs (consisting mainly of an increase of proliferating CD8). Glofitamab treatment further increased the frequency of monocytes and DCs in patients with CRs, and of NKs in both CRs and PDs. Within the DC population, we observed a significant decrease of cDC1 population and increase of the AXL+SIGLEC6+ DC population with high capability of driving T cell activation and proliferation upon treatment in both CR and PD patients. Complementary to changes in cell proportions, we identified gene expression programs associated with response, of which some were shared between two or more cell types while others were cell type-specific. Using established gene expression programs of naiveness, cytotoxicity, and exhaustion, we found that CD8 T cells of CR patients maintain their naive phenotype on treatment, whereas the naiveness of CD8 T cells of PD patients decreases upon treatment. Subsequently, we observed a higher naiveness of CD8 T cells in patients with CRs compared to PDs on treatment. Moreover, CD4 T cells increase their naiveness upon treatment in patients with CRs and decrease in patients with PDs, resulting in higher overall naiveness of CD4 T cells on treatment in CR patients compared to PD patients. In line with the expected mode of action, glofitamab treatment led to an increase of the cytotoxicity and exhaustion scores in CD8 T cells, and of the exhaustion score in CD4 T cells, of both CR and PD patients, reflective of T cell engagement in both patient groups. However, CD8 T cells of PD patients display overall a higher cytotoxicity and exhaustion level compared to CR patients. TCR sequencing data showed a higher clonal diversity in CD4 T cells compared to CD8 T cellsand and enrichment of hyper-expanded clonotypes in the CD8 T cell compared to CD4 T cell compartment . However, we neither detected an increase in the proportion of clonal cells, nor a change in clonal diversity upon treatment, regardless of the patient response group. In addition, we did not find any association between the percent of TCR repertoire occupied by clones of a given size and response or treatment time point. In agreement with the molecular analysis, T cells from CR patients (collected at baseline and on treatment) displayed a tendency towards higher cytotoxicity, cytokine secretion and T cell activation compared to PD patients upon ex vivo re-stimulation with glofitamab, suggestive of a better functional state of T cells from patients with CR compared to PD. Taken together, our work provides a thorough characterization of peripheral blood immune cells from CR and PD patients treated with glofitamab, and provides evidence that T cells from CR patients maintain a fresher/naive and more functional state compared to PD patients, which display T cells in a more advanced differentiation state with lower functionality.

Real-World Study of Polatuzumab Vedotin-Based Regimen for Relapsed or Refractory Diffuse Large B-Cell Lymphoma in Chinese Cohort: Updated Results of 2-Year Follow-up

Introduction Patients with relapsed or refractory (r/r) DLBCL had poor prognosis when ineligible to autologous hematopoietic stem cell transplantation (auto-HSCT). Polatuzumab vedotin (Pola) has demonstrated significant efficacy and acceptable safety in such patients in Clinical trials and real-world data. Here we reported long-term follow-up result of Pola-based regimen in real-world Chinese cohort. Aims To evaluate the efficacy and safety of Pola-based regimens for the treatment of r/r DLBCL using real-world data. Methods This retrospective study analyzed data from the Chinese Pola compassionate use program, conducted at four tertiary hospitals from December 2019 to June 2021. Patients with r/r DLBCL were enrolled. The treatment regimens included Pola in combination with rituximab (Pola+R) or with additional bendamustine (Pola+BR) administered for six cycles, with each cycle lasting 21 days. The objective response rate (ORR), progression-free survival (PFS), overall survival (OS) and adverse events (AEs) were analyzed. Results Thirty-nine patients were included in the analysis, with a median follow-up of 23.5 months (range: 0.6-35.2). The ORR at the end of treatment was 38.5% (15/39), while the best ORR achieved was 69.2% (27/39). The median PFS and OS were 6.6 months (95%CI: 3.7-9.6) and 15.6 months (95%CI: 3.4-27.9), respectively. Compared to those who did not complete six cycles of treatment, a significantly longer PFS (12.1 vs. 3.5 months, P=0.006) and OS (18.8 vs. 6.3 months, P=0.001) was observed in patients who completed all six cycles of treatment. Among 16 patients receiving Pola+R and 23 receiving Pola+BR, the proportion of patients who completed all six-treatment cycles was 56.3% and 34.8%, respectively. Grade 3-4 AEs were reported in 48.7% of patients with most commonly occurred being neutropenia (28.2%), leucopenia (17.9%) and thrombocytopenia (15.4%). Conclusions The 2-year follow-up analysis demonstrated sustained progression-free and overall survival, with no new safety signals. The full cycles of Pola-based regimen may contribute to better efficacy for real-world populations. These findings supported Pola-based regimens as an effective real-world treatment option for Chinese patients with r/r DLBCL. Key words Diffuse large B-cell lymphoma; refractory/relapsing; polatuzumab vedotin; real-world study; China.

Investigating the Drug Resistance Mechanisms of Refractory Diffuse Large B-Cell Lymphoma Based on Proteomic Research

Diffuse large B-cell lymphoma (DLBCL) is the most common histological subtype of non-Hodgkin's lymphoma, more than half of the patients of which can achieve complete remission or even be cured with multidrug immunochemotherapy regimen(R-CHOP). However, for patients with relapsed or refractory DLBCL, there is no effective treatment, while the unfavorable prognosis is related to the primary and acquired resistance to existing therapies. Proteomics is the study of the presence and activity of all proteins in cells. In exploring the mechanisms of DLBCL progression, proteomic technology can identify and quantify changed proteins in DLBCL, reveal the relationship between changes in protein expression levels and different stages of lymphoma occurrence and development, and thus provide information on variation in related proteins and protein-related signaling molecules. Based on proteomic technology research, this study is expected to discover new molecular mechanisms, further clarify resistance mechanisms, and find new therapeutic targets for DLBCL, providing more powerful weapons for better prognosis. We first collected paired clinical patient samples that met the experimental requirements and selected biopsy specimens from 3 patients who relapsed within 1 year after systemic treatment, including biopsy specimens at initial diagnosis and relapse. Next, paraffin sections and HE-stains were performed on the samples, and target areas were micro dissected using a laser microscope. The dissected samples were collected in specific EP tubes, lysed ultrasonically by adding lysis buffer to each group of samples, and the protein concentration of the supernatant was determined after centrifugation. Equal amounts of protein from each sample were digested with trypsin and the final concentration was controlled. Analysis was then performed using the EASY-nLC 1200 ultra-high performance liquid phase system and the Q Exactive TM HF-X mass spectrometer. Finally, bioinformatic analysis was performed to analyze differential proteins and find relevant drug targets or markers. The number of identified peptides was 36,107, the number of identified proteins was 6,338, and the number of quantifiable proteins was 6,165. The top five differentially upregulated proteins in the volcano plot of differential proteins were HOPX, TSNAX, PTMA, FTH1, and IFI44, respectively; and the top five downregulated differential proteins were KRT9, KRT1, KRT10, KRT2, and PIP, respectively. Through enrichment analysis of differential proteins, we noticed the ferroptosis pathway as a key pathway, and found that FTH1, TF, and GSH were involved in iron death-related processes, while FTH1 was one of the top five upregulated differential proteins. Iron death is an iron-dependent, non-apoptotic form of cell death discovered in recent years, activation of which can lead to tumor cell death and inhibit tumor growth. Increased expression of the iron-binding subunit FTH1 can suppress activation of iron death and reduce iron death in tumor cells. This suggests that by developing targeted drugs that act on increasing FTH1 expression or iron death inducers which are capable of slowing down the process, it may be possible to address the problem of traditional chemo-resistance in DLBCL and provide new directions for overcoming DLBCL resistance.

Risk of Bleeding and Thrombosis with CAR T-Cell Therapy: A Scoping Review

Introduction: Chimeric antigen receptor (CAR) T-cell therapy is emerging as a novel and revolutionary therapy in the treatment of relapsed or refractory lymphoma and other hematological malignancies. Patients with hematological malignancies are at risk of bleeding and thrombosis due to multiple risks factors. As there is an ongoing exploration of the incidence and characteristics of bleeding and thrombosis after CAR T-cell therapy, there is no existing published data that incorporates both peer-reviewed and non-peer-reviewed literature. Aim: Assess the risk of bleeding and thrombosis in CAR T-cell recipients for hematological malignancies. Method: We conducted a systematic literature search in MEDLINE, EMBASE, and CENTRAL electronic databases from inception to October 2022 using a combination of subject headings and text words. We included studies that assessed thrombotic and bleeding complications in patients eighteen or older who underwent CAR T-cell therapy for a hematologic malignancy. Our search strategy yielded 14 studies in the form of abstracts and full-text articles. The review process consisted of two levels of screening: (1) a title and abstract review and (2) a full-text review. Result: Of 3354 records, we included 14 studies based on our inclusion criteria [figure 1]. Majorities of the studies were retrospective. Most of the patients in these studies received CAR T-cell therapy for refractory diffuse large B-cell lymphoma. Patients received at least 2 lines of therapy or more before CAR T-cell treatment. Different types of CAR T-cell therapy were used. Based on the included studies, the risk of bleeding was higher than thrombosis [figure 2]. Bleeding sites were variable, but gastrointestinal (GI) bleeding was predominant. In Ma et al, GI bleeding occurred in 11 (42.3%) patients. In Qi et al and Johnsrud et al, bleeding occurred in 44 (11%) and 12 (9.4%) patients respectively. Variable sites of thrombosis were noticed. In Hashmi et al incidence of thrombosis was 10.8%. Although there are some risk factors associated with bleeding and thrombosis in this population, the exact mechanism is still unknown. We think future studies to determine the exact mechanism of this toxicity and to assess the underlying risk factors would be crucial to decrease morbidity and mortality in this population and to develop risk assessment tools.

A Multicenter, Open-Label, Phase 1b/2 Study to Evaluate the Effects of Maplirpacept in Combination with Glofitamab in People with Relapsed or Refractory Diffuse Large B Cell Lymphoma

Background Maplirpacept (PF-07901801) is an anti-CD47 fusion protein constructed to enhance phagocytosis and antitumor activity via inhibition of CD47-mediated signaling. Maplirpacept is currently being investigated in an ongoing Phase 1, multi-center, 1a/1b study in participants with advanced hematologic malignancies (NCT03530683). Early safety and efficacy results for maplirpacept monotherapy have previously been reported in patients with refractory or relapsed lymphomas (Patel et al. Blood 2021;138:3560). Study Design This trial is a multicenter, open-label, Phase 1b/2 study (NCT05896163), to evaluate the effects of maplirpacept in combination with fixed doses of glofitamab, after a single dose of obinutuzumab, in patients with relapsed or refractory diffuse large B-cell lymphoma (DLBCL). The estimated enrollment is 70 patients; the study will be split into 2 parts (Figure 1). Phase 1b will determine the maximum tolerated dose of maplirpacept, with fixed doses of glofitamab and obinutuzumab. In Phase 2, participants will be randomized to low or high doses of maplirpacept, with fixed doses of glofitamab and obinutuzumab, consistent with the FDA ‘Project Optimus’ initiative for dose optimization and selection in oncology drug development. Study interventions will be given intravenously in 21-day cycles. At Cycle 0, participants will receive a single pre-treatment dose of obinutuzumab followed by 2 step-up doses of glofitamab. The combination of maplirpacept with full dose glofitamab will be given at Cycle 1 Day 1. Maplirpacept will be given weekly for the first 3 cycles, then every 3 weeks. Glofitamab will be given every 3 weeks for up to 11 cycles. Subsequently, participants will continue to receive maplirpacept alone. In Phase 1b, participants will be allocated to sequential doses of maplirpacept, given with fixed doses of glofitamab, after 1 dose of obinutuzumab, to select doses of maplirpacept for further evaluation. The primary endpoint for Phase 1b is to assess dose limiting toxicities up to 21 days following the first dose. In Phase 2, participants will be randomized to 2 different doses of maplirpacept, administered with fixed doses of glofitamab, after 1 dose of obinutuzumab. The primary endpoint for Phase 2 is objective response as per the Lugano 2014 response criteria. Secondary endpoints include frequency of adverse events and clinical laboratory abnormalities, objective response (Phase 1b only), duration of response, complete response, duration of complete response, progression free survival, serum concentration vs time for maplirpacept and glofitamab, and immunogenicity of maplirpacept and glofitamab. Adult patients with histologically confirmed relapsed or refractory DLBCL (including DLBCL NOS, primary mediastinal large B cell lymphoma and transformed follicular lymphoma, T cell/histiocyte-rich large B-cell lymphoma, Grade 3b follicular lymphoma or composite lymphoma with a DLBCL component with a subsequent DLBCL relapse), will be eligible (Table 1). Participants with histological transformation to DLBCL from an earlier diagnosis of low-grade lymphoma are eligible, except for participants with history of Waldenström macroglobulinemia. Treatment with at least 1 prior line of anti-CD20 antibody-based therapy will be included. For Phase 2, no more than 2 prior lines of systemic therapy will be permitted. Patients who are not candidates for, or who are unwilling to undergo high dose chemotherapy and subsequent stem cell transplant and/or are unable or unwilling to receive chimeric antigen receptor T-cell therapy (e.g. for logistical reasons) will be eligible. All patients should have adequate bone marrow, hepatic, and renal function (eGFR ≥45 mL/min) and ECOG performance status ≤2. Exclusion criteria include prior treatment with an anti-CD47 agent, prior anti-CD20xCD3 containing regimen, refractoriness to an obinutuzumab monotherapy containing regimen, prior allogeneic stem cell transplantation or autologous stem cell transplantation within 12 weeks prior to enrolment, high grade B-cell lymphoma with BCL2 and Myc rearrangement, active bacterial, viral, fungal, mycobacterial, parasitic, or other infection.

Efficacy and Safety of Glofitamab in Patients with R/R DLBCL in Real Life Setting - a Retrospective Study

Introduction - Glofitamab is a CD20-directed CD3 T-cell engager that has recently been approved by the FDA for relapsed and refractory diffuse large B cell lymphoma (R/R DLBCL). The approval was based on a pivotal phase II study including 154 patients with a median of 3 lines of treatment (a third post chimeric antigen receptor T (CAR-T) cell therapy) demonstrating an overall response rate (ORR) and complete response (CR) rate of 52% and 39%, respectively 1. However real-life data is still lacking. The present study was aimed to determine the efficacy and safety of glofitamab in real-life settings. Methods -A retrospective multicenter study including consecutive patients aged 18 years and older with R/R DLBCL, treated through a national compassionate program with at least 1 dose of glofitamab, after failing at least three lines of therapy. The first patient was recruited in September 2020 till January 2023. Outcomes included ORR and CR, defined by the Lugano criteria; progression free survival (PFS) and overall survival (OS), defined from the day of obinutuzimab infusion until event; cytokine release syndrome (CRS) and immune effector cells-associated neurotoxicity syndrome (ICANS) were defined by the ASTCT consensus 2019. Risk factors for PFS and OS were identified by univariate and multivariate analyses, using logistic regression. Results - A total of 35 patients treated in 6 Israeli centers, were included. Median age at the time of initiation of glofitamab was 67 years (59-74) and 66% of the patients were males. The median number of previous therapies was 5 (IQR, 4-6) and 83% received more than 3 previous lines of therapy. 43% were primary refractory, all but 3 received CAR-T cell therapy (91%) and 23% underwent autologous stem cell transplantation (Table 1). Median follow up was 34 months (IQR, 18-57). Outcomes: 34% achieved ORR with 14% achieving CR. Median PFS and OS was 2 (IQR, 1-6) and 4 months (IQR, 2-10), respectively (Figure 1). At the end of follow up - 12 (34%) patients survived, 21 died and 2 patients were lost to follow- up. Causes of death included disease progression in 15 patients and infections in 6. Treatment was discontinued earlier than 12 cycles in 86%, mostly due to progressive disease (76%), and 24% due to infections (3 - COVID-19; 3- bacterial). Adverse events included CRS in 37% - all grade 1-2. 2 patients developed grade 1 ICANS. In a multivariate analysis including age, male, bulky disease, elevated LDH and CRS, only male sex was found to be significant risk factor for PFS and OS (p=0.02, p=0.04 respectively). Conclusions - In this real-life cohort of R/R DLBCL, glofitamab showed less efficacy than in the pivotal study. This might be due to the more heavily pretreated patients in our series with nearly all patients post CAR-T. Nevertheless, toxicity in our real-life analysis was better than reported in the pivotal study, supporting the role of glofitamab in R/R DLBCL patients. References- 1. Dickinson et al. Glofitamab for Relapsed or Refractory Diffuse Large B-Cell Lymphoma. NEJM 2022;387:2220-31