Refractory Diffuse Large B Cell Lymphoma Patients Research Articles

Combining Obinutuzumab With Radiation for Refractory DLBCL: Retrospective Safety and Efficacy Analysis

IntroductionApproximately 30% of patients with diffuse large B cell lymphoma (DLBCL) will develop relapsed or treatment refractory disease following primary chemotherapy. Patients unable to undergo aggressive chemotherapy and stem cell transplant (SCT) or CAR T-cell therapy have limited treatment options. Here, we investigated the safety and efficacy of combining Obinutuzumab with cytoreductive radiation to all areas of disease in patients with relapsed DLBCL. MethodsA retrospective review of patients with treatment refractory DLBCL was performed. All patients were treated with external beam radiation to all sites of refractory disease with concurrent and adjuvant Obinutuzumab. Toxicities were evaluated based on Common Terminology Criteria for Adverse Events (CTCAE) v5.0 criteria. Kaplan Meier analysis was utilized to calculate progression free survival (PFS) and overall survival (OS). ResultsBetween 2016 and 2022, 7 patients with refractory DLBCL were treated with concurrent radiation and Obinutuzumab. No grade 3 or greater treatment related toxicity was observed. Four of the seven patients had a complete response at the radiated site on first post-radiation imaging. The median PFS and OS were 30 months. ConclusionIn this small cohort of treatment refractory DLBCL patients, the combination of radiation and Obinutuzumab was well tolerated without excessive treatment related toxicity. The combination resulted in durable disease control with a prolonged overall survival without additional treatment in a subset of patients.

Unraveling the enigma of B cells in diffuse large B-cell lymphoma: unveiling cancer stem cell-like B cell subpopulation at single-cell resolution.

Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent form of aggressive non-Hodgkin lymphoma. Despite receiving standard treatment, a subset of patients undergoes refractory or recurrent cases, wherein the involvement of cancer stem cells (CSCs) could be significant. We comprehensively characterized B cell subpopulations using single-cell RNA sequencing data from three DLBCL samples and one normal lymph tissue. The CopyKat R package was employed to assess the malignancy of B cell subpopulations based on chromosomal copy number variations. CIBERSORTx software was utilized to estimate the proportions of B cell subpopulations in 230 DLBCL tissues. Furthermore, we employed the pySCENIC to identify key transcription factors that regulate the functionality of B cell subpopulations. By employing CellphoneDB, we elucidated the interplay among tumor microenvironment components within the B cell subpopulations. Finally, we validated our findings through immunofluorescence experiments. Our analysis revealed a specific cancer stem cell-like B cell subpopulation exhibiting self-renewal and multilineage differentiation capabilities based on the exploration of B cell subpopulations in DLBCL and normal lymph tissues at the single-cell level. Notably, a high infiltration of cancer stem cell-like B cells correlated with a poor prognosis, potentially due to immune evasion mediated by low expression of major histocompatibility complex molecules. Furthermore, we identified key transcription factor regulatory networks regulated by HMGB3, SAP30, and E2F8, which likely played crucial roles in the functional characterization of the cancer stem cell-like B cell subpopulation. The existence of cancer stem cell-like B cells in DLBCL was validated through immunofluorescent staining. Finally, cell communication between B cells and tumor-infiltrating T cell subgroups provided further insights into the functional characterization of the cancer stem cell-like B cell subpopulation. Our research provides a systematic description of a specific cancer stem cell-like B cell subpopulation associated with a poor prognosis in DLBCL. This study enhances our understanding of CSCs and identifies potential therapeutic targets for refractory or recurrent DLBCL patients.

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.

Selinexor in Combination with Salvage Regimen for Refractory Diffuse Large B-Cell Lymphoma (DLBCL) Patients with TP53 Alterations

Background TP53 has been identified as one of the most frequently altered gene in diffuse large B-cell lymphoma (DLBCL). The prognosis of DLBCL patients with TP53 mutations ( TP53mut) was poor prognosis in R-CHOP era, even with treatments like autologous stem-cell transplantation (ASCT) or anti-CD19 chimeric antigen receptor T-cell (CAR-T) therapy. Moreover, TP53 mutations are more prevalent at relapse and are associated with therapeutic resistance. Selinexor is a selective inhibitor of nuclear export with promising anti-cancer properties and preclinical studies have shown its potential to suppress tumor cells with TP53 mutations. Several clinical studies proved selinexor in combination with R-GDP/R-ICE/R-Gemox was effective in relapsed/refractory DLBCL. Method The study enrolled relapsed/refractory DLBCL patients with TP53 alterations (including TP53 mutation and/or deletion) for treatment with selinexor in combination with salvage regimens (R-GDP/R-ICE/R-Gemox) since November 2021 in our center. The treatment was administered orally as 40 mg of selinexor on days 1, 8, and 15, combined with one of the following regimens: R-GDP (R 375mg/m2 d0, gemcitabine 1g/m2*d1, cisplatin 25mg/m2 d1-d3, dexamethasone 40mg d1-d4). R-ICE (R 375mg/m2 d0, ifosfamide 1.5g/m2*3d, carboplatin (AUC 5) and etoposide 100mg/m2*3d). R-Gemox ((R 375mg/m2 d0, gemcitabine 1g/m2*d1, oxaliplatin 100mg/m2*d1) every 3 weeks as per physician's choice. Objective response rate (ORR) was evaluated every 3 cycles based on Lugano 2014 criteria and Adverse events (AE) were rated according to the NCI CTCAE 5.0. Results 20 patients were treated with selinexor plus salvage regimen in this study and received at least two cycles of treatment, making them eligible for analysis. The median age of the 20 patients was 54 years (range 15 to 77), of this patients, 12 (60%) were male, 12 (60%) patients had disease stage III-IV at screening, and 45% with IPI>2. The median number of prior regimen was 1 (range, 1-4) with 2 patients (2 L) and 1 patient (3L). ALL patients were refractory to the last line of treatment. The patients received a median of three cycles (range, 2-6) of selinexor 40mg once a week combined with salvage regimen (13 patients received selinexor plus R-GDP, 6 with selinexor plus R-ICE and 1 with selinexor plus R-Gemox). The mutational profile of patients was presented in Figure A (14 patients with TP53 mutation, 5 patients with TP53 mutation and deletion, 1 with TP53 deletion). The best overall and complete response rates were 75% and 25%, respectively. Among them, 6 patients in PR and 1 patient in SD received CART treatment, and 5 achieved PR and 2 with CR after CART treatment (Figure B). One patient experienced PD after selinexor+R-GDP and died of disease progression within one month after CART transfusion. 1 SD patient transferred to CD3/CD20 bispecific antibody clinical trial and still progressed during the bispecific treatment. Taken together, selinexor-based salvage regimen had promising response rate in refractory DLBCL with TP53 alterations, and could be a potential bridging approach before CART therapy. Patients responded to selinexor-based regimen tended to have better outcome after CART or bispecific antibody treatment. Conclusions TP53 alterations are associated with poor outcomes in DLBCL. We identified an effective dosing schedule of selinexor (40mg QW) in combination with R-GDP/R-ICE/R-Gemox for TP53-altered refractory DLBCL patients. Further research is warranted to investigate the mechanism of action behind this treatment approach

YAP1-Dependent Promotion of Downstream Transcription through BRD4 Mediates Selinexor Resistance in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent subtype of non-Hodgkin lymphoma. Despite advancements in treatment, the prognosis for relapsed and/or refractory DLBCL (R/R DLBCL) patients remains unfavorable due to the development of drug resistance and disease progression. The pressing need to enhance the outcomes in R/R DLBCL patients necessitates ongoing investigations into novel therapeutic approaches. Among various exportins, the nuclear export protein exportin 1 (XPO1) solely governs the transportation of nuclear proteins harboring leucine-rich hydrophobic nuclear export signals (NES), including essential regulators like p53, RB1, and p27. Selinexor, an oral inhibitor specifically targeting XPO1, has emerged as a promising candidate in R/R DLBCL patients. Encouraging findings from a phase II clinical trial demonstrate a more favorable efficacy of Selinexor in the germinal center B-cell (GCB) subtype compared with that in other subtypes of DLBCL. Nonetheless, the clinical implementation of Selinexor has led to the emergence of acquired resistance, posing a significant challenge for medical practitioners. Efforts to overcome this resistance and establish strategies to maximize Selinexor's therapeutic potential remain of paramount importance in addressing the urgent clinical needs of R/R DLBCL patients. Methods: Characterization of Selinexor resistance targets was performed using a CRISPR/Cas9 screen transcription factors library pool at 0-day and 14-day time points after treatment. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. Synergistic treatment approach utilizing BRD4 inhibitor JQ1, known for its curative effects in AML, MM, and ALL, in combination with Selinexor were also evaluated. Mechanistic exploration of BRD4's role in Selinexor resistance was further analyzed via RNA-seq, ChIP-seq, as well as luciferase reporter experiments. Therapeutic outcomes were evaluated in R/R DLBCL patients undergoing Selinexor treatment via PET/CT imaging. Results ： BRD4, BRD7, ZMYND8, and YTHDC1 were identified as top Selinexor resistance candidate targets through CRISPR/Cas9 Screen. The knock-down of BRD4 enhanced the killing ability of Selinexor in vitro and reduced tumor burden in subcutaneously transplanted tumor mice after treatment with Selinexor in vivo in DLBCL. The combined treatment with BRD4 inhibitor JQ1 and Selinexor had a stronger ability to promote cell apoptosis, enhance DNA damage and reduce nascent DNA compared with the single drug group. Hematological and organ toxicity tests confirmed an acceptable toxicity for the combined drug regimen. ChIP-seq differential peaks and RNA-seq differential genes analysis elucidated the crucial role of YAP1's sub-cellular localization under Selinexor treatment. YAP1 nuclear translocation enhanced transcription and formed an interaction with BRD4, resulting in augmented BRD4 binding domain, and finally lead to the resistance of Selinexor. PET/CT imaging showed that one patient with high XPO1 expression achieved complete response (CR), exhibiting a sensitivity to Selinexor treatment, but the other exhibits resistance associated with a lack of XPO1 expression and BRD4-regulated acetylation. Conclusions: The nuclear translocation of YAP1 and its interaction with BRD4 mediated the transcriptional regulation and further induced resistance to Selinexor in DLBCL. BRD4 inhibitor JQ1 combined with low-dose Selinexor can mitigate blood toxicity and enhance therapeutic efficacy, achieving an improved outcome.

Combining Obinutuzumab with Radiation for Treatment Refractory Diffuse Large B Cell Lymphoma: Retrospective Safety and Efficacy Analysis.

Approximately 30% of patients with diffuse large B cell lymphoma (DLBCL) will develop relapsed or treatment refractory disease following primary chemotherapy. Patients unable to undergo aggressive chemotherapy and stem cell transplant or CAR T-cell therapy have limited treatment options. Obinutuzumab, a type II CD-20 directed antibody, has been previously shown to have efficacy in these patients, though overall outcomes remain poor. Here, we investigated the safety and efficacy of combining obinutuzumab with cytoreductive radiation to all areas of disease in patients with relapsed DLBCL. A retrospective review of patients with treatment refractory DLBCL was performed. All patients were treated with external beam radiation to all sites of refractory disease with concurrent and adjuvant obinutuzumab. Patients were treated with obinutuzumab 1600 mg on days 1 and 8 of cycle 1, followed by 800 mg on day 1 for 8 total cycles or until progression. Toxicities were evaluated based on Common Terminology Criteria for Adverse Events (CTCAE) v5.0 criteria. Kaplan Meier analysis was utilized to calculate progression free survival (PFS) and overall survival (OS). Between 2016 and 2022, 8 patients with refractory DLBCL were treated with concurrent radiation and obinutuzumab. The median age at time of treatment was 70. The most common radiation dose was 50 Gy in 25 fractions (range 37.5 - 50 Gy). No grade 3 or greater treatment related toxicity was observed. The median number of prior lymphoma treatments was 2 (range 1-5). Fifty percent of patients had a complete response at the irradiated site, and no patients progressed at the site of radiation. Median PFS and OS were 30 months, with two patients dying of unrelated disease without recurrence at 30 and 60 months. Four patients did not develop evidence of recurrence with only one patient receiving additional therapy (planned CAR-T following radiation completion). In this small cohort of treatment refractory DLBCL patients, the combination of radiation and obinutuzumab was well tolerated without excessive treatment related toxicity. The combination resulted in durable disease control with prolonged overall survival without additional treatment in a subset of patients. Larger studies of this combination therapy in refractory DLBCL are warranted as bridging or definitive treatment.

Real-world experiences of CNS-directed chemotherapy followed by autologous stem cell transplantation for secondary CNS involvement in relapsed or refractory diffuse large B-cell lymphoma.

Secondary central nervous system (CNS) involvement is a rare but fatal event in patients with diffuse large B cell lymphoma (DLBCL). Some studies have suggested autologous stem cell transplantation (ASCT) for patients responding to salvage therapies, although its role is not clear. We analyzed DLBCL patients with secondary CNS involvement who received salvage therapies with curative intent and who underwent high-dose chemotherapy followed by ASCT. We analyzed the post-ASCT outcome in terms of CNS and/or systemic relapse and overall survival (OS) according to type of secondary CNS involvement and salvage treatment. A total of 43 patients who achieved complete or partial response after salvage treatments, mainly high-dose methotrexate (MTX)-containing chemotherapy, was treated with busulphan-thiotepa followed by ASCT between 2009 to 2019. Fifteen patients experienced grade III/IV febrile neutropenia, but all adverse events were manageable. At the median follow-up of 14.7 months after ASCT, 17 patients did not relapse, however, 26 patients had relapsed, comprising isolated CNS relapse (n = 12), systemic relapse (n = 12), and both (n = 2). Patients with systemic relapse had significantly shorter OS than those with isolated CNS relapse (42.7 vs, 11.1 months, p = 0.002). Of the 26 patients who relapsed after ASCT, six patients were rescued by subsequent salvage treatments. Finally, 21 patients were alive at the time of analysis. In conclusion, consolidative ASCT might be beneficial for secondary CNS involvement in relapsed or refractory DLBCL patients if they responded to CNS-directed salvage chemotherapy and were eligible for transplantation.

106P Zanubrutinib in combination with tislelizumab in patients with refractory diffuse large B cell lymphoma (DLBCL): A phase II study

Treatment of refractory DLBCL still represents a unique area of unmet need. Previous studies have shown synergistic antitumour effects between Bruton’s tyrosine kinase inhibitors (BTKi) and immune checkpoint inhibitors. However, it lacks data of zanubrutinib and tislelizumab. Herein, we present the premilitary results of an ongoing, multicenter, single-arm phase II study designed to assess the safety and efficacy of zanubrutinib in combination with tislelizumab in refractory DLBCL patients.

Outcomes in Refractory Diffuse Large B-Cell Lymphoma: Results from Two Prospective Korean Cohorts.

Diffuse large B-cell lymphoma (DLBCL) is the most common hematologic malignancy worldwide. Although substantial improvement has been achieved by the frontline rituximab-based chemoimmunotherapy, up to 40%-50% of patients will eventually have relapsed or refractory disease, whose prognosis is extremely dismal. We have carried out two prospective cohort studies that include over 1,500 DLBCL patients treated with rituximab plus CHOP (#NCT01202448 and #NCT02474550). In the current report, we describe the outcomes of refractory DLBCL patients. Patients were defined to have refractory DLBCL if they met one of the followings, not achieving at least partial response after 4 or more cycles of R-CHOP; not achieving at least partial response after 2 or more cycles of salvage therapy; progressive disease within 12 months after autologous stem cell transplantation. Among 1,581 patients, a total of 260 patients met the criteria for the refractory disease after a median time to progression of 9.1 months. The objective response rate of salvage treatment was 26.4%, and the complete response rate was 9.6%. The median overall survival (OS) was 7.5 months (95% confidence interval, 6.4 to 8.6), and the 2-year survival rate was 22.1%±2.8%. The median OS for each refractory category was not significantly different (p=0.529). In line with the previous studies, the outcomes of refractory DLBCL patients were extremely poor, which necessitates novel approaches for this population.

CYCLON and NPM1 Cooperate within an Oncogenic Network Predictive of R-CHOP Response in DLBCL.

Simple SummaryCYCLON is a nuclear protein, which has been associated with disease progression and treatment resistance in DLBCL, the most common form of aggressive B-cell lymphoma, but also represents a predictive factor of refractory disease and relapse for immuno-chemotherapy-treated DLBCL patients. The molecular mechanisms related to this unstructured protein remain largely uncharacterized. Here, we performed a mass-spectrometry-based identification of the CYCLON protein interactome that suggested it could exert nucleolar functions related to cell proliferation. Among the CYCLON oncogenic network, we performed an immunohistochemical evaluation of the multi-functional nucleolar protein NPM1 in a DLBCL cohort and showed that CYCLON/NPM1 concomitant expression delineates a poor prognosis subgroup of patients. Multivariate survival analyses demonstrated that specific sub-cellular localizations of CYCLON and NPM1 represent independent novel predictors specifically associated with refractory DLBCL.R-CHOP immuno-chemotherapy significantly improved clinical management of diffuse large B-cell lymphoma (DLBCL). However, 30–40% of DLBCL patients still present a refractory disease or relapse. Most of the prognostic markers identified to date fail to accurately stratify high-risk DLBCL patients. We have previously shown that the nuclear protein CYCLON is associated with DLBCL disease progression and resistance to anti-CD20 immunotherapy in preclinical models. We also recently reported that it also represents a potent predictor of refractory disease and relapse in a retrospective DLBCL cohort. However, only sparse data are available to predict the potential biological role of CYCLON and how it might exert its adverse effects on lymphoma cells. Here, we characterized the protein interaction network of CYCLON, connecting this protein to the nucleolus, RNA processing, MYC signaling and cell cycle progression. Among this network, NPM1, a nucleolar multi-functional protein frequently deregulated in cancer, emerged as another potential target related to treatment resistance in DLBCL. Immunohistochemistry evaluation of CYCLON and NPM1 revealed that their co-expression is strongly related to inferior prognosis in DLBCL. More specifically, alternative sub-cellular localizations of the proteins (extra-nucleolar CYCLON and pan-cellular NPM1) represent independent predictive factors specifically associated to R-CHOP refractory DLBCL patients, which could allow them to be orientated towards risk-adapted or novel targeted therapies.

Safety of Anti-CD-19 Chimeric Antigen Receptor T-Cell Therapy in the Older Population with Diffuse Large B Cell Lymphoma: A Meta-Analysis

IntroductionPatients with refractory or relapsed diffuse large B cell lymphoma (DLBCL) have very limited treatment options. Axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene maraleucel are the Anti-CD-19 chimeric antigen receptor T-cell (CAR-T) therapy have been recently approved for refractory or relapsed DLBCL patients who have failed at least two lines of treatment. CAR-T therapy has significant adverse effects, most notably cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), limiting its use, particularly in the elderly population. In this meta-analysis, we analyzed the safety of CAR-T therapy in the elderly population compared to the younger patients with DLBCL.MethodsRandomized controlled trials and observational studies in adult patients with DLBCL treated with anti-CD-19 CAR-T therapy were included in the meta-analysis. Case reports, case series, and review articles were excluded. We searched PubMed for studies published before 1st July 2021 using keywords “tisagenlecleucel,” “axicabtagene ciloleucel,” and “lisocabtagene maraleucel.” The study selection process is shown in figure 1. Statistical analysis was performed with Comprehensive meta-analysis version 3. The fixed-effect model was used for pooled analysis. Heterogeneity across studies was analyzed using I2 statistics.ResultsThree studies, comprising of 616 patients, were included in the analysis. Characteristics of the study population and the number of events in each study are shown in figure 2. Two studies used a cut-off of 65 years, while one study used a cut-off of 70 years for the older population. The mean age in the younger population was 55, while the mean age in the elderly population ranged from 69 to 76. Mean follow-up ranged widely in the three studies ranging from 7 months to 27.1 months.Incidence of CRS ranged from 36% to 84% in the elderly population compared to 34% to 83% in younger patients. Grade 3 or 4 CRS ranged from 1 to 14% in the elder compared to 2 to 14% in younger patients. The pooled analysis shown in figure 3 revealed an odd ratio of 1.11 with a confidence interval ranging from 0.76 to 1.63 (p=0.58), implying there is no statistical difference between the two groups.The incidence of ICANS ranged from 27% to 65% in elderly patients compared to 8.5% to 47% in younger patients. Grade 3 or 4 ICANS ranging from 2 to 47% in elderly patients compared to 2% to 18% in younger patients. The pooled analysis shown in figure 4 showed an odd ratio of 1.59 with a confidence interval ranging from 1.09 to 2.34 (p=0.02), suggesting the risk of ICANS is higher in older patients.ConclusionCRS and ICANS are the most common adverse effects limiting the use of CAR-T therapy in the elderly. Our analysis showed that age does not impact the risk of CRS in DLBCL patients treated with anti-CD19 CAR-T treatment. However, older patients are at a higher risk of ICANS as compared to younger, suggesting CAR-T therapy should be used with caution in patients older than 65 years of age, especially if they have baseline neurological impairment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Investigation of the Heterogeneity Evolution and Drug Resistance Mechanism of Diffuse Large B-Cell Lymphoma Under the Treatment of a Novel HDAC Inhibitor LAQ824

Aims:Diffuse large B-cell lymphoma (DLBCL), the most frequent malignant lymphoma subtype, is a group of highly invasive diseases with great heterogeneity in genomic alterations, clinical characteristics, morphological manifestations, treatment response and prognosis. Although most DLBCL patients can be cured by immunochemotherapy, nearly 40% of DLBCL patients still develop drug resistance and relapse. For relapsed/ refractory (R/R) DLBCL patients, there is still no optimal treatment. The heterogeneity and clonal evolution of tumor cells are the core driving forces for the occurrence and development of DLBCL, and the root causes for their refractory, recurrence and drug resistance. In this study, we screened out a novel small molecule compound effectively killing DLBCL cells, and analyzed its potential mechanism of anti-tumor. Meanwhile, by using single cell sequencing technology, we try to further investigate the heterogeneity and clona evolution and drug resistance mechanism of DLBCL under different drug pressure, explore core driver factors of drug resistance, evaluate and develop new treatment strategies.Methods:In this study, GEXSCOPE microfluidic platform was used for single-cell transcriptome sequencing. Seruat software was used for cell type recognition and clustering analysis. In order to further investigate the molecular mechanism of LAQ824 inducing the apoptosis of DLBCL cells and explore the target of LAQ824, antibody chip was performed to detect the phosphorylation of related signaling pathway. Gene expression was detected by real-time qPCR and Western blot. ChIP, RNA interfering (RNAi) and dual-luciferase activity assay were performed to validate the potential drug resistance targets for LAQ824. Moreover, WES of 21 DLBCL cell lines were performed to map mutations and analyze the correlation between related mutations and LAQ824 resistance. In this study, we established DLBCL animal models using NOD SCID mice transplanted with DLBCL cell lines, by which we could evaluate the tumor inhibition efficiency of LAQ824 alone and/or combination with other small molecular inhibitors.Results:Using GDSC database, we screened out Dacinostat (LAQ824), a novel HDAC inhibitor, was highly sensitive that could effectively induce the apoptosis of most DLBCL cells at low concentrations. Functional assay showed that LAQ824 could inhibit cell proliferation and promote apoptosis of tumor cells. LAQ824 treatment could significantly upregulate the acetylation level of histone H3 within a certain concentration range, and the DNA damage repair function of DLBCL cells was supressed by inhibiting Chk2 expression, thus significantly inducing cell apoptosis and effectively killing DLBCL cells. Meanwhile, through single-cell sequencing analysis, it was found that c-Fos could be activated under certain drug pressure of LAQ824. As a potential drug-resistant core driver gene, the expression level of c-Fos is highly correlated with IC50 of LAQ824 and the prognosis of patients with DLBCL, which can be used as a sensitivity indicator of LAQ824. Treatment with c-Fos inhibitor combined with LAQ824 can significantly improve the tumor inhibition rate, validated both in vitro and in vivo, which is expected to alleviate the recurrence and drug resistance of DLBCL patients.Conclusions:In general, we explores potential therapeutic drugs for DLBCL parients, adjusts and explores new clinical treatment strategies on this basis, and provides theoretical basis and data support for the realization of individualized precise treatment and the solution of DLBCL recurrence and drug resistance. DisclosuresNo relevant conflicts of interest to declare.

Immunotherapies Old and New: Hematopoietic Stem Cell Transplant, Chimeric Antigen Receptor T Cells, and Bispecific Antibodies for the Treatment of Relapsed/Refractory Diffuse Large B Cell Lymphoma.

Diffuse large B cell lymphoma (DLBCL) is curable in a majority of patients; however, a significant portion of patients develop relapsed or refractory disease. High-dose chemotherapy followed by autologous stem cell transplant is the standard approach in appropriately selected patients. Many patients are not candidates for transplant and many who do receive autologous transplant relapse. Therapies which harness T cells including chimeric antigen receptor T cells (CAR-T) and bispecific antibodies are active in this chemotherapy-resistant population. We review the role of autologous and allogeneic stem cell transplant, CAR-T therapy, and bispecific antibodies in the treatment of relapsed or refractory DLBCL. Phase I studies of bispecific antibodies directed against CD20 × CD3 have shown activity in heavily pre-treated DLBCL including in patients who have progressed following autologous transplant and/or CAR-T therapy. Two CAR-T products have received regulatory approval in relapsed or refractory DLBCL, with other products in clinical trials. CAR-T treatment has resulted in durable remissions and trials are ongoing to determine if CAR-T should replace autologous transplant as second-line therapy for DLBCL. The development of multiple T cell-directed therapies for DLBCL offers new treatment options for chemotherapy-resistant disease. We discuss our approach to relapsed or refractory DLBCL patients and the open question of optimal sequencing of autologous transplant (a current standard treatment), CAR-T therapy (FDA approved), and bispecific antibodies (in clinical trials).

Serial surveillance by circulating tumor DNA profiling after chimeric antigen receptor T therapy for the guidance of r/r diffuse large B cell lymphoma precise treatment.

Background: Circulating tumor DNA (ctDNA) released from tumor cells carries the tumor-associated genetic and epigenetic characteristics of cancer patients. Next-generation sequencing (NGS) facilitates the application of ctDNA profiling for identification and monitoring of minimal residual disease (MRD) in cancer, and can serve as the guidance for precise treatment.Methods: In this study, we profiled genomic alterations in the baseline, relapsed, and progressive tumor samples of eight diffuse large B cell lymphoma (DLBCL) patients (NCT03118180) after chimeric antigen receptor T (CAR-T) cell therapy.Results: The median follow-up was 41 months. 4 (50%) patients achieved complete remission (CR), 1 (12.5%) patient achieved partial remission (PR), and the other 3 (37.5%) patients showed no response. 3 of 5 patients who achieved remission relapsed within 4 months after CAR-T therapy, while the rest 2 patients remained CR for more than 3 years. Based on the positron emission tomography-computed tomography (PET-CT) scan, the current gold standard for evaluating response to therapy in lymphoma, the sensitivity and specificity of our ctDNA profiling in detecting tumor-related ctDNA mutations were 94.7% and 83.3%, respectively. The median numbers of baseline plasma ctDNA mutations in patients who remained long-term CR and patients who relapsed or became refractory to CAR-T therapy were 3 and 14.3, respectively. GNA13, SOCS1, TNFAIP3 and XPO1 mutations appeared to be associated with poor prognosis after CAR-T cell therapy. Our results also suggested that lenalidomide might relieve relapsed lymphoma with mutations in NFKBIA 202C>T (p.Q68*) and NFKBIE 433A>T (p.K145*) by targeting NF-Kappa B signaling. In addition, the inhibitor selinexor may be another choice for refractory or relapse (r/r) DLBCL patients after CAR-T cell treatment.Conclusion: Serial ctDNA monitoring is an emerging technology for the surveillance of disease status and prognosis prediction. In this work, we demonstrated the use of serial ctDNA monitoring in r/r DLBCL patients after CD19-targeted CAR-T cell therapy. Our longitudinal NGS profiling revealed the changes of ctDNA mutation in accordance with prognosis, and shed some light on exploring more targeted treatment options together with CAR-T cell therapy.

High-dose rituximab in combination with autologous stem cell transplantation for relapsed or refractory diffuse large B cell lymphoma

ObjectivesThe aim of this study was to evaluate the efficacy and toxicity of high-dose rituximab (HD-R) in combination with autologous stem cell transplantation (auto-SCT) in patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL). MethodsThere were 22 patients in the HD-R group, to whom rituximab was administered during stem cell mobilization (375mg/m2 1 day before and 7 days after chemotherapy) and after transplantation (1000mg/m2 on days +1 and +8). In the control group, the procedure was the same as that in the HD-R group but without rituximab. We observed the safety, tolerability, adverse effects and immune reconstitution of HD-R therapy. The log-rank test, univariate analysis and multivariate Cox regression analysis were used to evaluate the effect of HD-R on survival. ResultsIn total, 22 relapsed or refractory DLBCL patients were treated with HD-R. No dose-limiting toxicities were observed except for CD19+ B cell reconstruction in the first 6 months after SCT. There were 20 relapsed or refractory DLBCL patients in the control group. The 3-year progression-free survival (PFS) and overall survival (OS) greatly improved in the HD-R group compared to that in the control group (63.8% vs. 35.0%, P=0.028 and 80.1% vs. 50.0%, P=0.035, respectively). The univariate and multivariate analyses demonstrated that HD-R and the time to relapse were independent prognostic factors for OS and PFS. ConclusionHD-R in combination with auto-SCT is a feasible and promising treatment for patients with relapsed or refractory DLBCL.

Hematopoietic stem cell transplantation and chimeric antigen receptor Tcell for relapsed or refractory diffuse large B-cell lymphoma.

Aim: Autologous hematopoietic stem cell transplantation (ASCT) is the standard-of-care curative treatment for relapsed or refractory diffuse large B-cell lymphoma (RR-DLBCL), but the relapse rate is usually high. Materials & methods: In this study, we treated 14 RR-DLBCL patients by combining ASCT and anti-CD19 chimeric antigen receptorT-cell therapy. Results: Eleven(78.57%) patients achieved completeor partialremission. Median duration of progression-free survivalwas 14.82months (95% CI: 0.00-31.20months) with 6-month progression-free survival rate of 64.29% (95% CI: 39.18-89.40%). Median overall survivalwas not achieved, with 1-year overall survival rate of 65.48% (95% CI: 36.00-94.96%). No neurotoxicity was observed. Conclusion: Our study demonstrated safety and feasibility of ASCT and anti-CD19 chimeric antigen receptorT-cell treatment for RR-DLBCL patients.

High-dose rituximab in combination with autologous stem cell transplantation for relapsed or refractory diffuse large B cell lymphoma

ObjectivesThe aim of this study was to evaluate the efficacy and toxicity of high-dose rituximab (HD-R) in combination with autologous stem cell transplantation (auto-SCT) in patients with relapsed or refractory diffuse large B cell lymphoma (DLBCL). MethodsThere were 22 patients in the HD-R group, to whom rituximab was administered during stem cell mobilization (375mg/m2 1 day before and 7 days after chemotherapy) and after transplantation (1000mg/m2 on days +1 and +8). In the control group, the procedure was the same as that in the HD-R group but without rituximab. We observed the safety, tolerability, adverse effects and immune reconstitution of HD-R therapy. The log-rank test, univariate analysis and multivariate Cox regression analysis were used to evaluate the effect of HD-R on survival. ResultsIn total, 22 relapsed or refractory DLBCL patients were treated with HD-R. No dose-limiting toxicities were observed except for CD19+ B cell reconstruction in the first 6 months after SCT. There were 20 relapsed or refractory DLBCL patients in the control group. The 3-year progression-free survival (PFS) and overall survival (OS) greatly improved in the HD-R group compared to that in the control group (63.8% vs. 35.0%, P=0.028 and 80.1% vs. 50.0%, P=0.035, respectively). The univariate and multivariate analyses demonstrated that HD-R and the time to relapse were independent prognostic factors for OS and PFS. ConclusionHD-R in combination with auto-SCT is a feasible and promising treatment for patients with relapsed or refractory DLBCL.

Selective delivery of T22-PE24-H6 to CXCR4+ diffuse large B-cell lymphoma cells leads to wide therapeutic index in a disseminated mouse model.

Background: Novel therapeutic strategies are urgently needed to reduce relapse rates and enhance survival in Diffuse Large B-Cell Lymphoma (DLBCL) patients. CXCR4-overexpressing cancer cells are good targets for therapy because of their association with dissemination and relapse in R-CHOP treated DLBCL patients. Immunotoxins that incorporate bacterial toxins are potentially effective in treating haematological neoplasias, but show a narrow therapeutic index due to the induction of severe side effects. Therefore, when considering the delivery of these toxins as cancer therapeutics, there is a need not only to increase their uptake in the target cancer cells, and their stability in blood, but also to reduce their systemic toxicity. We have developed a therapeutic nanostructured protein T22-PE24-H6 that incorporates exotoxin A from Pseudomonas aeruginosa, which selectively targets lymphoma cells because of its specific interaction with a highly overexpressed CXCR4 receptor (CXCR4+) in DLBCL.Methods: T22-PE24-H6 cytotoxicity and its dependence on the CXCR4 receptor were evaluated in DLBCL cell lines using cell viability assays. Different in vitro experiments (mitochondrial membrane potential, Western Blot, Annexin V and DAPI staining) were conducted to determine T22-PE24-H6 cell death mechanisms. In vivo imaging and therapeutic effect studies were performed in a disseminated DLBCL mouse model that mimics organ infiltration in DLBCL patients. Finally, immunohistochemistry and histopathology analyses were used to evaluate the antineoplastic effect and systemic toxicity.Results: In vitro, T22-PE24-H6 induced selective cell death of CXCR4+ DLBCL cells by activating the apoptotic pathway. In addition, repeated T22-PE24-H6 intravenous administration in a CXCR4+ DLBCL-disseminated mouse model showed a significant reduction of lymphoma burden in organs clinically affected by DLBCL cells (lymph nodes and bone marrow). Finally, we did not observe systemic toxicity associated to the nanoparticle treatment in non-DLBCL-infiltrated organs.Conclusion: We have demonstrated here a potent T22-PE24-H6 antineoplastic effect, especially in blocking dissemination in a CXCR4+ DLBCL model without associated toxicity. Thereby, T22-PE24-H6 promises to become an effective alternative to treat CXCR4+ disseminated refractory or relapsed DLBCL patients.

Highly Favorable Outcomes with Salvage Radiation Therapy Followed By Autologous Transplant in Relapsed and Refractory DLBCL Patients with Minimal or No Response to Salvage Chemotherapy

Introduction: For patients with relapsed or primary refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL) who respond to salvage chemotherapy, high-dose chemotherapy and autologous hematopoietic cell transplantation (HDT-AHCT) is considered standard of care. Patients with refractory disease to salvage chemotherapy, defined as stable disease (SD) or progressive disease (PD), by functional imaging are ineligible for HDT-AHCT, and have a poor prognosis. In practice, we have attempted to salvage these patients with radiation therapy (RT) to residual sites of active disease prior to consolidative HDT-AHCT. The outcome of this unique combined modality salvage paradigm has not been previously reported. Methods: We retrospectively reviewed all patients with rel/ref DLBCL who received salvage chemotherapy followed by salvage RT and HDT-AHCT between the years of 2000 and 2017 at a single center. Only patients with SD or PD as defined on the 5-point Deauville scale after salvage chemotherapy and who had at least 1 year of follow-up were included in this analysis. The second-line age-adjusted International Prognostic Index (sAAIPI) was determined at the time of initiation of salvage chemotherapy.Survival functions were estimated by the Kaplan-Meier method and compared using a log-rank test. Results: Thirty-six patients, 12 with relapsed and 24 with primary refractory disease, with a median age of 44 years (range: 19-68 years) were analyzed. Twenty-three patients had DLBCL while 13 had primary mediastinal B-cell lymphoma (PMBCL). The majority of patients had KPS 80-100 (n=32, 89%), 0-1 extranodal sites (n=30, 83%), and normal LDH (n=21, 58%). The sAAIPI scores for this cohort were as follows: 0 (n=10), 1 (n=21), 2 (n=4), and 3 (n=1). All patients received salvage chemotherapy with subsequent functional imaging showing SD (n=32) and PD (n=4) and then went on to receive salvage RT to the sites of active disease. Median RT dose was 39.6Gy (range: 30-54Gy). Six patients also received TBI as part of their conditioning regimen prior to HDT-AHCT. With median follow up of 4.0 years (range: 1.0-12.3 years) for survivors, 4-year relapse-free survival (RFS) was 75.6% and 4-year overall survival (OS) was 80.3% (Figure 1a). There was no significant difference in 4-year RFS for patients with relapsed versus primary refractory disease (80.2% vs 74.8%, p=0.59). PMBCL patients had better RFS than DLBCL patients (92.3% vs 67.8%, p=0.12). Using the composite sAAIPI score was highly prognostic with worse outcomes for patients with higher risk sAAIPI scores. By sAAIPI score, 4-year RFS was 80.0% for a score of 0, 90.2% for a score of 1, and 0% for scores of 2 and 3. Patients with low- and low-intermediate risk sAAIPI scores of 0 and 1 had improved RFS as compared to patients with sAAIPI scores of 2 and 3 (87.0% vs 0%, p<0.0001 (Figure 1b). Conclusions: Patients with chemorefractory rel/ref DLBCL who have had minimal or no response to systemic salvage therapy may benefit from salvage RT to the residual PET-avid disease followed by HDT-AHCT, particularly if their sAAIPI score is ≤ 1. The outcome of this retrospective cohort is markedly superior to outcomes described in the literature for this high-risk population and represents a promising treatment paradigm to be further explored. Emerging data suggest similar patients may benefit from CAR T-cell therapy. Given the limited availability and high cost of CAR T-cell therapy, we suggest there may be a role for sequencing this combined-modality salvage paradigm prior to CAR T-cell therapy in order to provide these poor-risk patients with an additional line of therapy. Disclosures Scordo: Angiocrine Bioscience, Inc.: Consultancy; McKinsey & Company: Consultancy. Sauter:Sanofi-Genzyme: Consultancy, Research Funding; GSK: Consultancy; Spectrum Pharmaceuticals: Consultancy; Novartis: Consultancy; Genmab: Consultancy; Precision Biosciences: Consultancy; Kite/Gilead: Consultancy; Celgene: Consultancy; Juno Therapeutics: Consultancy, Research Funding.

PD-1 Inhibitor Combined with Chemotherapy As a Salvage Therapy for Refractory DLBCL

Purpose: Tostudy the efficacy and side effects of programmed death 1 (PD-1) inhibitor combined with chemotherapy for refractory diffuse large B cell lymphoma (DLBCL). Methods: Six refractory DLBCL patients with very high tumor burden or progresses rapidly disease were enrolled in a clinical trial that Toripalimab in combination with second-line chemotherapy at the Department of Hematology in Tianjin First Center Hospital (Tianjin, China). All of the eligible patients had received another clinical trial of anti-CD19 chimeric antigen receptor (CAR) modified T-cell therapy before. They had ineffective to anti-CD19 CAR-T cell therapy or had effective ever but the disease progression again. The patients were received the following combination therapy. Toripalimab (240mg) administered on day -4 and received DHAP (Cisplatin: 100mg/m2 on day 1, Cytarabine: 2g/m2q12h on day 2, Dexamethasone: 40mg/day from day 1 to 4) every 3 weeks at the same dose. Or Toripalimab administered on day 0 and received GemOx (Gemcitabine: 1000mg/m2 on day 1 and 8, Oxaliplatin: 130mg/m2 on day 1) every 3 weeks. The DNA level of anti-CD19 CAR gene in peripheral blood was detected by quantitative polymerase chain reaction (qPCR) analysis, in order to prove the PD-1 inhibitor had no combined effect with the CAR-T cells. Tumour imaging by CT or PET-CT was done at baseline, and after every course of treatment. The adverse events (AEs) were collected throughout the study and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (Version 4.0). Results: The median time from infusion to cutoff day was 6 months (Range: 1 to 6 months). After 2 weeks of the first course of combination treatment, all the six patients refractory DLBCL patients reached partail (PR) response of their disease. After the second or three courses of treatment, three patients kept their disease stable state for 5 months (Patient 1#), 3 months (Patient 4#) and 2 months (Patient 2#). The other three patients received only one course of treatment. One patient kept his disease stable state for 3 months (Patient 5#), one patient died from cerebrovascular disease 2 months after the combination treatment (Patient 6#). The last one patient received allogeneic haploid hematopoietic stem cell transplantation 1 months after the combination treatment (Patient 3#). No patient withdrew from the combination therapy because of treatment-related AEs. Furthermore, no treatment-related or immune-mediated deaths occurred in our study. The average proportion of anti-CD19 CAR-T cells and the mean DNA level of anti-CD19 CAR gene in peripheral blood maintained in very low levels throughout the combination therapy. It proved that the PD-1 inhibitor had no combined effect with the anti-CD19 CAR-T cells received before. Conclusions: The PD-1 inhibitor combined with second-line chemotherapy could be as a salvage therapy for refractory DLBCL without treatment-related AEs. Figure Disclosures No relevant conflicts of interest to declare.