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Loncastuximab tesirine (Lonca) and chemoimmunotherapy (CIT) have been assessed in patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), but direct evidence from head-to-head randomized clinical trials is not available.Matching-adjusted indirect comparison (MAIC) was used to evaluate the efficacy of Lonca versus CIT-era treatment in R/R DLBCL. The analysis used individual patient data from the phase II LOTIS-2 trial of Lonca (NCT03589469) and pooled aggregated data from 2 extension studies of the CORAL trial for CIT. The LOTIS-2 trial included 145 patients who had relapsed or progressed following 2 or more multi-agent systemic treatment regimens; the CORAL extension studies included 203 patients who received 2 prior lines of therapy and 75 patients who relapsed after autologous hematopoietic cell transplantation. MAIC analyses were performed to adjust for cross-trial differences in inclusion/exclusion criteria and the distribution of observed baseline characteristics. Overall response rate (ORR) and overall survival (OS) were compared between the balanced trial populations.A total of 80 patients in LOTIS-2 were included in the analysis. After matching to the characteristics of 278 patients from the pooled CORAL extension studies, the ORR was significantly higher for Lonca compared with CIT-era treatment (53.4% vs. 40.3%, P.05). Lonca was also associated with a significantly improved OS compared with CIT-era treatment (median OS 10.8 vs. 6.4 months; adjusted hazard ratio: 0.67 [95% CI: 0.48, 0.92], P.05).This study indicates that Lonca was associated with significantly improved efficacy compared with CIT-era treatments for R/R DLBCL.

Chimeric antigen receptor T-cell (CAR-T cell) therapy directed at CD19 produces durable remissions in the treatment of relapsed/refractory non-Hodgkin lymphoma (NHL). Nonetheless, many patients receiving CD19 CAR-T cells fail to respond for unknown reasons. To reveal changes in 4-1BB–based CD19 CAR-T cells and identify biomarkers of response, we used single-cell RNA sequencing and protein surface marker profiling of patient CAR-T cells pre- and postinfusion into patients with NHL. At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a nonproliferative, highly differentiated, and exhausted state, with an enriched exhaustion profile in CAR-T cells of patients with poor response marked by TIGIT expression. Utilizing in vitro and in vivo studies, we demonstrate that TIGIT blockade alone improves the antitumor function of CAR-T cells. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving a poor response in patients with NHL. Significance: This is the first study investigating the mechanisms linked to CAR-T patient responses based on the sequential analysis of manufactured and infused CAR-T cells using single-cell RNA and protein expression data. Furthermore, our findings are the first to demonstrate an improvement of CAR-T cell efficacy with TIGIT inhibition alone. This article is highlighted in the In This Issue feature, p. 1825

Supplementary Data from Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma

Chimeric antigen receptor T-cell (CAR-T cell) therapy directed at CD19 produces durable remissions in the treatment of relapsed/refractory non-Hodgkin lymphoma (NHL). Nonetheless, many patients receiving CD19 CAR-T cells fail to respond for unknown reasons. To reveal changes in 4-1BB–based CD19 CAR-T cells and identify biomarkers of response, we used single-cell RNA sequencing and protein surface marker profiling of patient CAR-T cells pre- and postinfusion into patients with NHL. At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a nonproliferative, highly differentiated, and exhausted state, with an enriched exhaustion profile in CAR-T cells of patients with poor response marked by TIGIT expression. Utilizing in vitro and in vivo studies, we demonstrate that TIGIT blockade alone improves the antitumor function of CAR-T cells. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving a poor response in patients with NHL.Significance:This is the first study investigating the mechanisms linked to CAR-T patient responses based on the sequential analysis of manufactured and infused CAR-T cells using single-cell RNA and protein expression data. Furthermore, our findings are the first to demonstrate an improvement of CAR-T cell efficacy with TIGIT inhibition alone.This article is highlighted in the In This Issue feature, p. 1825

Approximately, a third of patients with diffuse large B-cell lymphoma (DLBCL) have refractory or relapsed disease after initial treatment. Despite recent regulatory approval of several new agents, including CAR-T cell therapy, polatuzumab vedotin and tafasitamab, there is still a need for additional therapies that expand the therapeutic alternatives and improve outcomes for patients with DLBCL that progresses after first line therapy.Studies of recently approved agents for relapsed DLBCL are reviewed. The relevance of CD19 as an immunotherapeutic target. The pharmacologic composition of loncastuximab tesirine and its cytotoxic payload, a pyrrolobenzodiazepine dimer. Phase I and phase 2 data for loncastuximab tesirine in non-Hodgkin lymphoma, showing the safety profile of this drug and the emerging efficacy results in DLBCL.Loncastuximab tesirine is an antiCD19 antibody drug conjugate with a novel cytotoxic payload. Early studies showed this drug is tolerable, with a safety profile that is different from other antibody drug conjugates approved for lymphoid malignancies. Efficacy data shows activity in different non-Hodgkin lymphoma entities, and a phase 2 study has been completed in DLBCL showing durable responses, including in high-risk subgroups. Loncastuximab tesirine will be an important addition to the treatment alternatives for DLBCL.

Monoclonal antibody treatment initially heralded an era of molecularly targeted therapy in oncology and is now widely applied in modulating anti-cancer immunity by targeting programmed cell receptors (PD-1, PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and, more recently, lymphocyte-activation gene 3 (LAG3). Chimeric antigen receptor T-cell therapy (CAR-T) recently proved to be a valid approach to inducing anti-cancer immunity by directly modifying the host’s immune cells. However, such cell-based therapy requires extensive resources such as leukapheresis, ex vivo modification and expansion of cytotoxic T-cells and current Good Manufacturing Practice (cGMP) laboratories and presents significant logistical challenges. Bi-/trispecific antibody technology is a novel pharmaceutical approach to facilitate the engagement of effector immune cells to potentially multiple cancer epitopes, e.g., the recently approved blinatumomab. This opens the opportunity to develop ‘off-the-shelf’ anti-cancer agents that achieve similar and/or complementary anti-cancer effects as those of modified immune cell therapy. The majority of bi-/trispecific antibodies target the tumor-associated antigens (TAA) located on the extracellular surface of cancer cells. The extracellular antigens represent just a small percentage of known TAAs and are often associated with higher toxicities because some of them are expressed on normal cells (off-target toxicity). In contrast, the targeting of intracellular TAAs such as mutant RAS and TP53 may lead to fewer off-target toxicities while still achieving the desired antitumor efficacy (on-target toxicity). Here, we provide a comprehensive review on the emerging field of bi-/tri-specific T-cell engagers and potential therapeutic opportunities.

To investigate camidanlumab tesirine (Cami) exposure-response (E-R) relationships, using an integrated population pharmacokinetic model, for patients with classical Hodgkin lymphoma (cHL) and non-Hodgkin lymphoma enrolled in an open-label, phase 1 study (NCT02432235).Exploratory analyses investigated relationships between exposure measures (CExploratory analyses showed a significant, positive relationship between exposure and ORR/OS. The final model showed this effect was non-significant due to the covariate effects. Cami exposures were higher in patients with selected grade ≥ 2 AEs at cycle 6 (the anticipated steady-state exposure level), confirmed in the final E-R models.Based on univariate results, C

Ibrutinib is effective in the treatment of relapsed/refractory (R/R) marginal zone lymphoma (MZL) with an overall response rate (ORR) of 48%. However, factors associated with response (or lack thereof) to ibrutinib in R/R MZL in clinical practice are largely unknown. To answer this question, we performed a multicenter (25 US centers) cohort study and divided the study population into three groups: “ibrutinib responders”—patients who achieved complete or partial response (CR/PR) to ibrutinib; “stable disease (SD)”; and “primary progressors (PP)”—patients with progression of disease as their best response to ibrutinib. One hundred and nineteen patients met the eligibility criteria with 58%/17% ORR/CR, 29% with SD, and 13% with PP. The median PFS and OS were 29 and 71.4 months, respectively, with no difference in PFS or OS based on the ibrutinib line of therapy or type of therapy before ibrutinib. Patients with complex cytogenetics had an inferior PFS (HR = 3.08, 95% CI 1.23–7.67, p = 0.02), while those with both complex cytogenetics (HR = 3.00, 95% CI 1.03–8.68, p = 0.04) and PP (HR = 13.94, 95% CI 5.17–37.62, p p = 0.03). In this largest study to date evaluating outcomes of R/R MZL treated with ibrutinib, we show that patients with primary refractory disease and those with PP on ibrutinib are very high-risk subsets and need to be prioritized for experimental therapies.

There is an increased risk of congestive heart failure (CHF) following anthracycline-based chemotherapy in patients with Diffuse Large B-cell lymphoma (DLBCL). Little is known about risk factors of CHF, other cardiovascular events (CVE), and CVE effect on outcomes. We conducted a retrospective review of 463 newly diagnosed DLBCL patients treated between 2002 and 2016 with anthracycline containing regimens. At a median follow up of 71.3 months, 10.4% patients developed new CHF, 4.97% had new atrial fibrillation and 3.2% had new coronary artery disease. Age over 65, advanced stage DLBCL and diabetes were associated with increased cumulative incidence of CVE. Patients with prior diabetes had decreased progression-free survival and overall survival in comparison to non-diabetics. Patients who had a CVE in the first year had significant worse OS then patients who did not have a CVE (Hazard Ratio 10.0, 95% CI, 7.24-13.88). A risk score incorporating age at DLBCL diagnosis, baseline lymphocyte count, disease stage and diabetes stratified into groups with low, intermediate and high risk for CVE, with 1-year cumulative incidence of CVE of 5.3%, 7.9% and 13.4%. Diffuse large B-cell lymphoma patients treated with anthracycline containing regimens have high incidence of CVE, which are not limited to CHF. Clinical variables at the time of diagnosis can identify the group of DLBCL patients at highest risk of CVE, for whom preventive interventions should be considered.

Background: Chimeric antigen receptor T cell therapy (CAR T) has demonstrated remarkable clinical efficacy in hematological malignancies. However, compromised T cell effector function, proliferation, and persistence can limit CAR T from reaching their full curative potential. Interleukin-2 (IL-2) is a potent stimulator of T cells, however therapeutic use of IL-2 is limited by systemic toxicity due its pleiotropy. Therefore, to provide a selective IL-2 signal to engineered T cells, we have developed a human orthogonal ligand/receptor system consisting of a half-life extended pegylated IL-2 mutein (STK-009) and a mutated IL-2 Receptor Beta (hoRb) that responds to STK-009 but not wild type IL-2. SYNCAR-001 is an autologous CAR T co-expressing a CD19 CAR and hoRb on the T cell surface. In mouse models of refractory lymphoma, STK-009 treatment led to expansion and activation of SYNCAR-001 cells with a maintenance of stem cell memory and effector T cell phenotypes. When added to SYNCAR-001, STK-009 increased complete response rate and durable responses in a dose dependent manner. In non-human primate studies, STK-009 alone demonstrated no significant biological activity in IL-2 sensitive populations (T cells or NK cells) and was tolerable without toxicity. Methods: This is a first-in-human, open-label, dose escalation study of combination SYNCAR-001 + STK-009 in adults with relapsed or refractory (r/r) CD19+ hematologic malignancies. The objectives of this study are to evaluate the safety, preliminary efficacy, pharmacokinetics, immunogenicity, and pharmacodynamics of SYNCAR-001 + STK-009. Dose escalation follows a standard 3+3 design with STK-009 being escalated while SYNCAR-001 is held at a single fixed dose. A dose extension will enroll a cohort of patients treated at a selected dose level and indication based on dose escalation findings. SYNCAR-001 is dosed intravenously (IV) once at Day 0 and STK-009 is dosed subcutaneously (SC) over the course of the study. Participants receive a single safety lead-in dose of STK-009 prior to lymphodepletion and subsequent SYNCAR-001 infusion. After SYNCAR-001 initiation, STK-009 is dosed SC weekly for 12 weeks and then monthly for 3 months. Eligible participants include individuals with r/r chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and selected r/r B-cell non-Hodgkin lymphoma (NHL subtypes of large B cell, mantle cell, and indolent lymphoma). Prior CD19 CAR use is excluded. The primary endpoint of safety includes outcomes such as adverse events and dose-limiting toxicities. Secondary endpoints include assessments of response, pharmacokinetics, and immunogenicity. Exploratory endpoints include assessment of immune cell populations, and relevant gene/protein expression, as well as persistence, phenotype, and functionality of SYNCAR-001 in the peripheral blood and/or bone marrow in response to STK-009. Citation Format: Maria Lia Palomba, Matthew G. Mei, Paolo F. Caimi, Matthew Cortese, Marco Davila, Greg Yadnik, Nestor Huang, David Mou, Martin Oft, Paul-Joseph Aspuria, Anita Mehta-Damani, Navneet Ratti, Naiyer Rizvi, Alex Azrilevich, Ran Reshef. A Phase 1 study to evaluate the safety and tolerability of a combination autologous CD19 CAR T cell therapy (SYNCAR-001) and orthogonal IL-2 (STK-009) in subjects with relapsed or refractory CD19 expressing hematologic malignancies (NCT05665062) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT125.

The COVID-19 pandemic has had a distinct spatiotemporal pattern in the United States. Patients with cancer are at higher risk of severe complications from COVID-19, but it is not well known whether COVID-19 outcomes in this patient population were associated with geography.To quantify spatiotemporal variation in COVID-19 outcomes among patients with cancer.This registry-based retrospective cohort study included patients with a historical diagnosis of invasive malignant neoplasm and laboratory-confirmed SARS-CoV-2 infection between March and November 2020. Data were collected from cancer care delivery centers in the United States.Patient residence was categorized into 9 US census divisions. Cancer center characteristics included academic or community classification, rural-urban continuum code (RUCC), and social vulnerability index.The primary outcome was 30-day all-cause mortality. The secondary composite outcome consisted of receipt of mechanical ventilation, intensive care unit admission, and all-cause death. Multilevel mixed-effects models estimated associations of center-level and census division-level exposures with outcomes after adjustment for patient-level risk factors and quantified variation in adjusted outcomes across centers, census divisions, and calendar time.Data for 4749 patients (median [IQR] age, 66 [56-76] years; 2439 [51.4%] female individuals, 1079 [22.7%] non-Hispanic Black individuals, and 690 [14.5%] Hispanic individuals) were reported from 83 centers in the Northeast (1564 patients [32.9%]), Midwest (1638 [34.5%]), South (894 [18.8%]), and West (653 [13.8%]). After adjustment for patient characteristics, including month of COVID-19 diagnosis, estimated 30-day mortality rates ranged from 5.2% to 26.6% across centers. Patients from centers located in metropolitan areas with population less than 250 000 (RUCC 3) had lower odds of 30-day mortality compared with patients from centers in metropolitan areas with population at least 1 million (RUCC 1) (adjusted odds ratio [aOR], 0.31; 95% CI, 0.11-0.84). The type of center was not significantly associated with primary or secondary outcomes. There were no statistically significant differences in outcome rates across the 9 census divisions, but adjusted mortality rates significantly improved over time (eg, September to November vs March to May: aOR, 0.32; 95% CI, 0.17-0.58).In this registry-based cohort study, significant differences in COVID-19 outcomes across US census divisions were not observed. However, substantial heterogeneity in COVID-19 outcomes across cancer care delivery centers was found. Attention to implementing standardized guidelines for the care of patients with cancer and COVID-19 could improve outcomes for these vulnerable patients.

In the last decade, a better understanding of the molecular pathogenesis of B-cell non-Hodgkin lymphomas has resulted in the development of novel targeted therapies, such as small molecule inhibitors of select kinases in the B-cell receptor pathway, antibody-drug conjugates, and small molecules that target a variety of proteins (eg, CD-19, EZH2, and XPO-1-mediated nuclear export). Anti-CD19 CAR T-cell therapy, first approved for relapsed/refractory (R/R) diffuse large B-cell lymphoma, has also emerged as a novel treatment option for R/R follicular lymphoma and mantle cell lymphoma. These NCCN Guideline Insights highlight the new targeted therapy options included in the NCCN Guidelines for B-Cell Lymphomas for the treatment of R/R disease.

ImportanceDetermining whether neurofilament light chain (NfL) elevations in patients who develop immune effector cell–associated neurotoxicity syndrome (ICANS) occur before or after infusion of cellular product is important to identify high-risk patients and inform whether neuroaxonal injury is latent or a consequence of treatment.ObjectiveTo quantify serial NfL levels in patients undergoing cellular therapy.Design, Setting, and ParticipantsThis retrospective 2-center study examined plasma NfL levels in 30 patients with detailed medical and treatment history, including all major pretreatment and posttreatment risk factors. Exclusion criteria included dementia and severe, symptomatic central nervous system (CNS) involvement.Main Outcomes and MeasuresPatients’ NfL levels were measured at 7 time points: baseline (prelymphodepletion), during lymphodepletion, postinfusion day (D) 1, D3, D7, D14, and D30. Prediction accuracy for the development of ICANS was next modeled using receiver operating characteristic (ROC) classification. Finally, univariate and multivariate modeling examined the association between NfL levels, ICANS, and potential risk factors including demographic (age, sex), oncologic (tumor burden, history of CNS involvement), neurologic (history of nononcologic CNS disease or neuropathy), and neurotoxic exposure histories (vincristine, cytarabine, methotrexate, or CNS radiotherapy).ResultsA total of 30 patients (median [range] age, 64 [22-80] years; 12 women [40%] and 18 men [60%]) were included. Individuals who developed ICANS had elevations in NfL prior to lymphodepletion and chimeric antigen receptor T-cell infusion compared with those who did not develop ICANS (no ICANS: 29.4 pg/mL, vs any ICANS: 87.6 pg/mL; P Conclusions and RelevanceIn a subset of patients in this cross-sectional study, the risk of developing ICANS was associated with preexisting neuroaxonal injury that was quantifiable with plasma NfL level. This latent neuroaxonal injury was present prior to drug administration but was not associated with historic neurotoxic therapies or nononcologic neurologic disease. Preinfusion NfL may further permit early screening and identification of patients most at risk for ICANS. Additional studies are needed to determine NfL’s utility as a predictive biomarker for early (preemptive or prophylactic) intervention and to delineate the origin of this underlying neural injury.

Reduced-intensity conditioning (RIC) regimens frequently provide insufficient disease control in patients with high-risk hematologic malignancies undergoing allogeneic hematopoietic stem cell transplantation (HSCT). We evaluated intensification of fludarabine/busulfan (Flu/Bu) RIC with targeted marrow irradiation (TMI) in a dose escalation with expansion phase I clinical trial. TMI doses were delivered at 1.5 Gy in twice daily fractions on days -10 through -7 (dose levels: 3 Gy, 4.5 Gy, and 6 Gy), Flu (30 mg/m

Introduction: Variability in expression of B-cell surface antigens could be an important mechanism of treatment failure after immunotherapy in patients with relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL). Understanding the evolution of target antigen expression can guide selection and sequencing of newer therapies targeting CD19 and CD20. We sought to quantitate the change in expression of CD19 and CD20 in R/R DLBCL after Rituximab (R)-based chemo immunotherapy (CIT) relative to pretreatment levels. Methods: Pts. diagnosed with DLBCL after January 2014 who were R/R to R-based CIT (R-CHOP or R-EPOCH), were identified and clinical variables at diagnosis (Dx) and first R/R were recorded. Data from archival flow cytometry assays on Dx and R/R biopsies were accessed. Using FCS Express® software, neoplastic cells were gated, and fluorescence intensity (FI) of CD19 and CD20 expression were reported. Multivariate linear mixed model was used to compare median and geometric mean FI of CD19 and CD20 between Dx and R/R. Results: A total of 51 flow cytometry assays (26 Dx and 25 R/R) were analyzed for 33 pts., of whom paired assays (at both Dx and R/R) were available for 18 pts. Median age at Dx was 64 (range, 41-76) yrs, 24 pts. (73%) were male and 29 (88%) had IPI ≥ 2. Cell of origin at Dx was GCB in 16 (49%), non-GCB in 12 (36%) pts., while two had a double-hit rearrangement. Median time to R/R was 10.4 months. There was a significant reduction in median FI of CD20 from Dx [median: 40,610 (range: 167 - 259,962)] to R/R [median: 11,596 (range: 63 - 79,592)], representing a reduction of 63% at R/R relative to Dx (P= 0.01; 95% CI: 20-73%). Similar change was observed in geometric mean FI of CD20, which was reduced 65% at R/R relative to Dx (P< 0.01; 95%CI: 31-82%). Median and geometric mean FI of CD19 at R/R were 38% and 20% lower compared to Dx, respectively, but these differences were not statistically significant (P= 0.08 and 0.39, respectively). Relative change in FI at R/R in individual cases, compared to the mean FI of all Dx cases, showed that 21 out 25 R/R cases (84%) had reduction of CD20 whereas only 14/25 (56%) had reduction of CD19. Interestingly, 7/25 (28%) R/R cases had an increase in CD19 expression by >80%. Reduction in CD20 geometric mean FI from Dx to R/R was significantly associated with age >60 years (p=0.04), bone marrow involvement (p 1 site of extra nodal involvement (p= 0.03) at Dx. Conclusions: Quantitative assessment by flow cytometry revealed a significant decline in expression of CD20 at R/R compared to Dx in patients with DLBCL treated with R-based CIT. CD19 expression was largely unchanged, although dramatically upregulated in a subset of R/R cases. Given the role of CD19 mediated pathways in B-cell NHL and its association with PI3K pro-survival signaling, these data merit further exploration as a potential mechanism of treatment resistance. Citation Format: Agrima Mian, Narendra Bhattarai, Wei Wei, Manishkumar S. Patel, Paolo F. Caimi, Sarah L. Ondrejka, Brian T. Hill. Quantitative assessment of the evolution of therapeutic target antigen expression in diffuse large b-cell lymphoma in response to treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3480.

The mechanism of aging is not yet fully understood. It has been recognized that there are age-dependent changes in the DNA methylation pattern of the whole genome. To date, there are several DNA methylation-based estimators of the chronological age. A majority of the estimators use the DNA methylation data from a single tissue type, such as blood. In 2013, for the first time, Steve Horvath reported the DNA methylation-based age estimator (353 CpGs were used) that could be applied to multiple tissues. A refined, more sensitive version that uses 391 CpGs was subsequently developed and validated in human cells, including fibroblasts. In this review, the age predicted by DNA methylation-based age estimator is referred to as DNAmAge, and the biological process controlling the progression of DNAmAge is referred to as the epigenetic aging in this minireview. The concepts of DNAmAge and epigenetic aging provide us opportunities to discover previously unrecognized biological events controlling aging. In this article, we discuss the frequently asked questions regarding DNAmAge and the epigenetic aging by introducing recent studies of ours and others. We focus on addressing the following questions: (1) Is there any synchronization of DNAmAge between cells in a human body?, (2) Can we use in vitro (cell culture) systems to study the epigenetic aging?, (3) Is there an age limit of DNAmAge?, and (4) Is it possible to change the speed and direction of the epigenetic aging? We describe our current understandings to these questions and outline potential future directions. IMPACT STATEMENT: Aging is associated with DNA methylation (DNAm) changes. Recent advancement of the whole-genome DNAm analysis technology allowed scientists to develop DNAm-based age estimators. A majority of these estimators use DNAm data from a single tissue type such as blood. In 2013, a multi-tissue age estimator using DNAm pattern of 353 CpGs was developed by Steve Horvath. This estimator was named “epigenetic clock”, and the improved version using DNAm pattern of 391 CpGs was developed in 2018. The estimated age by epigenetic clock is named DNAmAge. DNAmAge can be used as a biomarker of aging predicting the risk of age-associated diseases and mortality. Although the DNAm-based age estimators were developed, the mechanism of epigenetic aging is still enigmatic. The biological significance of epigenetic aging is not well understood, either. This minireview discusses the current understanding of the mechanism of epigenetic aging and the future direction of aging research.

Chimeric antigen receptor T cells (CAR-T cell) targeting CD19 are effective against several subtypes of CD19-expressing hematologic malignancies. Centralized manufacturing has allowed rapid expansion of this cellular therapy, but it may be associated with treatment delays due to the required logistics. We hypothesized that point of care manufacturing of CAR-T cells on the automated CliniMACS Prodigy® device allows reproducible and fast delivery of cells for the treatment of patients with non-Hodgkin’s lymphoma. Here we describe cell manufacturing results and characterize the phenotype and effector function of CAR-T cells used in a phase I/II study. We utilized a lentiviral vector delivering a second-generation CD19 CAR construct with 4-1BB costimulatory domain and TNFRSF19 transmembrane domain. Our data highlight the successful generation of CAR-T cells at numbers sufficient for all patients treated, a shortened duration of production from twelve to eight days followed by fresh infusion into patients, and the detection of CAR-T cells in patient circulation up to one-year post-infusion.

Introduction: Patients with diffuse large B-cell lymphoma (DLBCL) that is resistant to chimeric antigen receptor T-cell (CAR-T) therapy have poor outcomes (Chow VA, et al. Am J Hematol. 2019;94:E209-E13). The majority of patients with DLBCL who relapse after CAR-T therapy do so with disease that continues to express CD19 surface antigen (Shah NN, Fry TJ. Nat Rev Clin Oncol. 2019;16:372-85); however, it is unknown whether treatment with CD19-targeted agents is an effective strategy for patients with prior failure of anti-CD19 CAR-T therapy. Loncastuximab tesirine (loncastuximab tesirine-lpyl; Lonca) is an FDA-approved CD19-directed antibody-drug conjugate (ADC) which had encouraging phase 1 antitumor activity and acceptable safety in non-Hodgkin lymphoma (Hamadani M, et al. Blood. 2021;137:2634-2645). In the Phase 2 LOTIS-2 trial (NCT03589469) the efficacy and safety of Lonca was evaluated in patients with relapsed or refractory (R/R) DLBCL after ≥2 lines of systemic treatments (Caimi PF, et al. Lancet Oncol. 2021;22:790-800). The overall response rate (ORR) was 48.3%. The aim of this post-hoc analysis of the LOTIS-2 trial was to investigate the antitumor activity of Lonca in patients with DLBCL relapsed or refractory after CAR-T therapy. Methods: The methodology of the LOTIS-2 trial has been published. Briefly, patients were treated with Lonca (0.15 mg/kg for the first 2 cycles then 0.075 mg/kg for subsequent cycles) administered as a single 30-minute infusion, once every 3 weeks for up to 1 year, or until progressive disease or unacceptable toxicity. Patients with previous anti-CD19 CAR-T therapy were required to have persistent CD19 expression, evaluated by local review of immunohistochemistry of a post-CAR-T biopsy. The primary endpoint was ORR, defined as the proportion of patients with best overall response of complete response (CR) or partial response (PR), determined by independent review. Secondary endpoints included overall survival (OS), progression free survival (PFS), and duration of response (DOR). PET/CT imaging was performed 6 and 12 weeks after the first Lonca dose and every 9 weeks thereafter. Response was assessed using the Lugano 2014 criteria. Kaplan Meier survival analysis was performed from initiation of Lonca treatment. Results: The characteristics of 13 patients with DLBCL with disease relapse or progression after anti-CD19 CAR-T therapy are shown in table 1. The median time interval between CAR-T infusion and Lonca treatment was 7 months (range, 45-400 days). Ten (77%) patients received Lonca as the first therapy after CAR-T failure, 3 patients received other treatments prior to Lonca (chemoimmunotherapy [R-GemOx], n = 1; allogenic stem cell transplant, n = 1; chemoimmunotherapy [R-GemOx] followed by venetoclax + bromodomain inhibitor, n =1). The ORR to Lonca was 46.2% (n=6; CR, 15.4% [n = 2]; PR, 30.8% [n = 4]) after a median of 2 cycles (range, 1-9). Of the 6 patients who achieved a response to Lonca, 5 had a previous response to CAR-T and 1 had prolonged, stable disease for >1 year after CAR-T. With a median follow-up of 8 months, the median OS and PFS were 8.2 and 1.4 months, respectively (Figure 1); the 1-year OS estimate was 33.3%. The median DOR to Lonca was 8 months. Conclusions: Lonca achieved a response in 6 out of 13 patients who had failed prior CAR-T therapy. Five out of 6 responding patients had previously presented at least a partial response after CAR-T therapy. These data suggest that in patients without CD19 antigen loss, repeat therapy with another agent targeting this antigen can result in disease control. Prior response to anti-CD19 therapy may be associated with subsequent response to a second anti-CD19 treatment. Further studies are needed to confirm the feasibility and value of repeated anti-CD19 treatments in patients with B-cell non-Hodgkin lymphoma. Funding: This study was funded by ADC Therapeutics; medical writing support was provided by CiTRUS Health Group. Figure 1 Figure 1. Disclosures Caimi: Amgen Therapeutics.: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); ADC Theraputics: Consultancy, Research Funding; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy; Verastem: Consultancy; Seattle Genetics: Consultancy; TG Therapeutics: Honoraria. Ardeshna: Gilead, Beigene, Celegene, Novartis and Roche: Honoraria; Norvartis, BMS, Autolus, ADCT, Pharmocyclics and Jansen: Research Funding; Gilead, Beigene, Celegene, Novartis and Roche: Membership on an entity's Board of Directors or advisory committees. Reid: Aptose Biosciences: Other: Serves as Principle Investigator, Research Funding; ADC Therapeutics: Other: Serves as Principle Investigator, Research Funding; Millennium Pharmaceuticals: Other: Serves as Principle Investigator, Research Funding; Xencor: Other: Serves as Principle Investigator, Research Funding. Ai: Kymria, Kite, ADC Therapeutics, BeiGene: Consultancy. Lunning: Myeloid Therapeutics: Consultancy; Spectrum: Consultancy; Daiichi-Sankyo: Consultancy; Verastem: Consultancy; Janssen: Consultancy; AstraZeneca: Consultancy; Morphosys: Consultancy; Beigene: Consultancy; Legend: Consultancy; ADC Therapeutics: Consultancy; Acrotech: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; AbbVie: Consultancy; Kite, a Gilead Company: Consultancy; TG Therapeutics: Consultancy; Novartis: Consultancy; Kyowa Kirin: Consultancy; Karyopharm: Consultancy. Zain: Secura Bio, DaichiSankyo, Abbvie: Research Funding; Kiyoaw Kirin, Secura Bio, Seattle Genetics: Honoraria; Secura Bio, Ono , Legend, Kiyowa Kirin, Myeloid Therapeutics Verastem Daichi Sankyo: Consultancy. Solh: ADCT Therapeutics: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy; BMS: Consultancy; Partner Therapeutics: Research Funding. Kahl: AbbVie, Acerta, ADCT, AstraZeneca, BeiGene, Genentech: Research Funding; AbbVie, Adaptive, ADCT, AstraZeneca, Bayer, BeiGene, Bristol-Myers Squibb, Celgene, Genentech, Incyte, Janssen, Karyopharm, Kite, MEI, Pharmacyclics, Roche, TG Therapeutics, and Teva: Consultancy. Hamadani: Takeda, Spectrum Pharmaceuticals and Astellas Pharma: Research Funding; Janssen, Incyte, ADC Therapeutics, Omeros, Morphosys, Kite: Consultancy; Sanofi, Genzyme, AstraZeneca, BeiGene: Speakers Bureau.

Introduction: There is increased risk of congestive heart failure (CHF) following anthracycline-based chemotherapy in patients with Diffuse Large B-cell lymphoma (DLBCL). Other than increased cumulative anthracycline dose, little is known about risk factors for developing CHF and other cardiovascular events (CVE). Methods: We conducted a retrospective analysis of 539 DLBCL patients diagnosed at University Hospitals Seidman Cancer Center between 2002 - 2016. Baseline patient and disease characteristics, patterns of treatment and outcomes after therapy, including response, survival, relapse, CVE, and causes of death were collected. Time-to-CVE was measured from the date of DLBCL diagnosis to the date of CVE diagnosis and censored at the date of last follow-up for those without CVE, considering death as competing risk. The cumulative incidence of CVE was estimated taking death as competing risk into account. Univariate and multivariable analysis on time-to-CVE was done using Gray's method. Multicollinearity was tested and some of the highly correlated covariables were excluded in the final analysis. CVE risk scores were computed for each patient as a linear combination of the factors included in the multivariable model and their respective coefficients. The optimal cutoff point was identified by searching the spectrum of the risk scores to have the best discrimination of time-to-CVE among patients at low/high risks. All tests were two-sided and p-value ≤ 0.05 were considered statistically significant. Results: We identified 463 patients who received anthracycline to be included in the statistical analysis. Baseline clinical and disease characteristics including comorbidities, clinical risk factors are outlined in Table 1. After a median follow up of 71.1 (range: 0.3 - 216.1) months, 5-year PFS was 59% with median PFS of 82.7 (95% Cl: 71.1 - 115.7) months and 5-year OS was 71% with median OS of 152.6 (95% CI: 127.5 - 162) months. CVE were identified in 92 patients (19.9%), with 128 events identified. The most common CVE was new onset of CHF in 10.4% of patients, followed by atrial fibrillation (4.9%) and asymptomatic heart failure ( 3%) (Table 2). The 3, 6 and 9-year cumulative incidence of CVE for early stage was 13.1%, 15.2% and 18.5%, respectively and 22.2%, 31.4% and 36.5% for advanced stage, respectively (p-value = 0.026). Similarly, the cumulative incidence of CVE was increased in patients with diabetes, with a 3-year cumulative incidence of CVE of 9.4% vs. 17.5% in patients with and without diabetes, respectively. Proportional hazards regression for time-to-CVE identified age >65 years, CD10 expression by IHC, diabetes, and hypertension as significant predictors of time-to-CVE on univariate analysis. On multivariable regression analysis with advanced age at diagnosis, stage, baseline lymphocyte count, and diabetes; stage was still significant in predicting time-to- CVE controlling the effects of advanced age and diabetes, which were also marginally significant. A predictive formula was created to help identify patients at highest risk for cumulative incidence of CVE. Using advanced age (over 65 years of age), baseline lymphocyte count above 800/µL, advanced stage at diagnosis and history of diabetes patients could be stratified into groups with low, intermediate and high risk for CVE (Table 4 and Figure 1). Risk score = Age >65 (3 points) + Lymphocyte count > 800/µL (-1 point) + Stage III/IV(8 points) + Diabetes (3 points) (Table 3) One-year cumulative incidence of CVE was 5.3% for low-risk patients, 7.9% for intermediate risk patients and 13.4% for high risk patients (Figure 1). Conclusions: Our analysis indicates a high risk of CVE in anthracycline treated DLBCL patients, with 19.9% of patients having a CVE in our cohort. The proposed CVE predictive score uses clinical and laboratory parameters routinely collected on all new DLBCL patients and can be easily assessed in the clinical setting. Patients in the high-risk group have a cumulative incidence of CVE of 13.4% at 1 year and would likely benefit from surveillance and interventions. Use of a clinical risk score can aid in the identification of patients at highest CVE risk to conduct studies of surveillance and prevention of this common complication. Figure 1 Figure 1. Disclosures Boughan: Beigene: Speakers Bureau. Caimi: Seattle Genetics: Consultancy; Verastem: Consultancy; Amgen Therapeutics.: Consultancy; Kite Pharmaceuticals: Consultancy; TG Therapeutics: Honoraria; ADC Theraputics: Consultancy, Research Funding; Genentech: Research Funding; XaTek: Patents & Royalties: Royalties from patents (wife).

Introduction: Loncastuximab tesirine (loncastuximab tesirine-lpyl; Lonca) is an FDA-approved CD19-directed antibody-drug conjugate (ADC) indicated for the treatment of adult patients with relapsed or refractory (R/R) large B-cell lymphoma after ≥2 lines of systemic therapy, including patients with high-grade B-cell lymphoma. In the Phase 2 LOTIS-2 trial (NCT03589469), Lonca was evaluated as a single agent in patients with R/R diffuse large B-cell lymphoma (DLBCL) and the overall response rate (ORR) was 48.3% (Caimi PF, et al. Lancet Oncol. 2021; 22:790-800). In an analysis of the 11 patients from the LOTIS-2 clinical trial with high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBCL; data cutoff: April 6, 2020), 45.5% (n=5) achieved an overall response. In this patient subgroup, the median duration of response was 13.37 months, and the median progression-free survival was 9.13 months. The aim of this analysis (data cut off: March 1, 2021) was to further characterize the clinical characteristics and efficacy of Lonca in patients with HGBCL enrolled in the LOTIS-2 clinical trial. Methods: The methodology for the LOTIS-2 trial has been published. Briefly, Lonca (0.15 mg/kg for the first 2 cycles followed by 0.075 mg/kg for subsequent cycles) was administered as a single 30-minute infusion, once every 3 weeks for up to 1 year, until progressive disease or unacceptable toxicity. The primary outcome was ORR defined as the proportion of patients with best overall response of complete response (CR) or partial response (PR), assessed by independent review. Investigator assessment of histopathology according to the 2016 WHO classification was used to define HGBCL. Results: The demographics and disease characteristics of the 11 patients with HGBCL are shown in Table 1. The median age was 74 years; 5 patients (45.5%) were age ≥75 years. The majority of patients received ≥3 prior lines of therapy (n = 8; 72.7%). One patient had a prior stem cell transplant (autologous; 9.1%). Of the 11 patients with HGBCL, the overall response rate was 45.5%, with all 5 patients achieving CR. The median (min, max) time to first CR or PR response was 43.0 (38, 148) days and the median time to first CR response was 79.0 (38, 148) days. All five responding patients (45.5%) had a duration of response ≥1 year; median duration of response has not been reached at the time of data cutoff. Conclusions: Efficacy data from this small subgroup of patients with HGBCL enrolled in the LOTIS-2 clinical trial are consistent with the overall trial population. These results suggest that Lonca is active in the treatment of this high-risk lymphoma subgroup, achieving comparable response rates with other risk groups and with long-term disease control in responding patients. Funding: This study was funded by ADC Therapeutics; medical writing support was provided by CiTRUS Health Group. Figure 1 Figure 1. Disclosures Alderuccio: Inovio Pharmaceuticals: Other: Family member; Agios Pharmaceuticals: Other: Family member; Puma Biotechnology: Other: Family member; ADC Therapeutics: Consultancy, Research Funding; Oncinfo / OncLive: Honoraria; Forma Therapeutics: Other: Family member. Ai: Kymria, Kite, ADC Therapeutics, BeiGene: Consultancy. Radford: ADC Therapeutics: Consultancy, Current holder of individual stocks in a privately-held company, Honoraria, Speakers Bureau; BMS: Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau; AstraZeneca: Current holder of individual stocks in a privately-held company. Solh: Jazz Pharmaceuticals: Consultancy; BMS: Consultancy; Partner Therapeutics: Research Funding; ADCT Therapeutics: Consultancy, Research Funding. Ardeshna: Gilead, Beigene, Celegene, Novartis and Roche: Honoraria; Norvartis, BMS, Autolus, ADCT, Pharmocyclics and Jansen: Research Funding; Gilead, Beigene, Celegene, Novartis and Roche: Membership on an entity's Board of Directors or advisory committees. Lunning: AbbVie: Consultancy; Novartis: Consultancy; Beigene: Consultancy; ADC Therapeutics: Consultancy; Daiichi-Sankyo: Consultancy; Spectrum: Consultancy; Acrotech: Consultancy; AstraZeneca: Consultancy; Myeloid Therapeutics: Consultancy; Janssen: Consultancy; Verastem: Consultancy; Celgene, a Bristol Myers Squibb Co.: Consultancy; Kite, a Gilead Company: Consultancy; Kyowa Kirin: Consultancy; Karyopharm: Consultancy; TG Therapeutics: Consultancy; Morphosys: Consultancy; Legend: Consultancy. Hess: BMS: Speakers Bureau; ADC Therapeutics: Consultancy. Zinzani: EUSAPHARMA: Consultancy, Other, Speakers Bureau; SANDOZ: Other: Advisory board; ROCHE: Other, Speakers Bureau; MSD: Consultancy, Other: Advisory board, Speakers Bureau; NOVARTIS: Consultancy, Other, Speakers Bureau; TG Therapeutics: Other: Advisory board, Speakers Bureau; KYOWA KIRIN: Other, Speakers Bureau; TAKEDA: Other: Advisory board, Speakers Bureau; Beigene: Other, Speakers Bureau; Incyte: Other, Speakers Bureau; ADC Therap.: Other; SERVIER: Other: Advisory board, Speakers Bureau; BMS: Other: Advisory board, Speakers Bureau; JANSSEN-CILAG: Other: Advisory board, Speakers Bureau; GILEAD: Other: Advisory board, Speakers Bureau; CELLTRION: Other: Advisory board, Speakers Bureau; VERASTEM: Consultancy, Other: Advisory board, Speakers Bureau. Stathis: Abbvie and PharmaMar: Other: travel grant; Pfizer, ADC Therapeutics, Bayer, Roche, Merck, Novartis, MEI Therapeutics and Abbvie: Research Funding; Bayer / Eli Lilly: Consultancy. Carlo-Stella: Sanofi: Consultancy, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Honoraria; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria; Janssen Oncology: Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Karyopharm Therapeutics: Membership on an entity's Board of Directors or advisory committees. Hamadani: Sanofi, Genzyme, AstraZeneca, BeiGene: Speakers Bureau; Janssen, Incyte, ADC Therapeutics, Omeros, Morphosys, Kite: Consultancy; Takeda, Spectrum Pharmaceuticals and Astellas Pharma: Research Funding. Kahl: AbbVie, Acerta, ADCT, AstraZeneca, BeiGene, Genentech: Research Funding; AbbVie, Adaptive, ADCT, AstraZeneca, Bayer, BeiGene, Bristol-Myers Squibb, Celgene, Genentech, Incyte, Janssen, Karyopharm, Kite, MEI, Pharmacyclics, Roche, TG Therapeutics, and Teva: Consultancy. Ungar: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Kilavuz: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yu: ADC Therapeutics: Current Employment. Qin: ADC Therapeutics: Current Employment, Current equity holder in publicly-traded company. Caimi: TG Therapeutics: Honoraria; Amgen Therapeutics.: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); ADC Theraputics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Verastem: Consultancy; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy.

Background: AntiCD19 CAR-T cells are effective against chemorefractory B cell lymphoma. Patients (pts) with rapidly progressive disease and urgent need for therapy have very poor prognosis and may not be able to receive CAR-T cells in time. Decreasing the apheresis to infusion time can make CAR-T cells rapidly available. We conducted a dual-center phase I trial using on-site manufacture of CAR-T cells for treatment of relapsed and refractory (r/r) B cell lymphoma. Methods: Adult pts with r/r CD19+ B cell lymphomas who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc, LTG1563) encoding an antiCD19 binding motif, CD8 linker, TNFRS19 transmembrane region, and 4-lBB/CD3z intracellular signaling domains. GMP-compliant manufacture was done using CliniMACS Prodigy in a 12-day culture, subsequently shortened to 8 days. Dose escalation was done using 3+3 design. Lymphodepletion included cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) were graded using the Lee and CARTOX criteria, respectively. CAR-T persistence was measured with qPCR and flow cytometry. Plasma cytokine concentrations were measured using electrochemiluminescence (MesoScale Diagnostics, Inc). Results: Thirty-one pts were enrolled and treated. Baseline patient and disease characteristics are listed in table 1. Twenty-nine (94%) pts were refractory to the prior line of therapy and 21 (68%) had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 45% [range 15-66], median culture expansion was 36-fold [range 3-79]. CAR-T cell doses were 0.5 x 10 6/kg (n = 4), 1 x 10 6/kg (n = 16), and 2 x 10 6/kg (n = 11). Median time from apheresis to lymphodepletion was 7 days (range 2 - 15) and median time from apheresis to CAR-T cell infusion time was 13 days (range 9 - 20). Twenty-eight pts were infused fresh product. Seventeen pts (55%) experienced CRS. Grade 1-2 CRS was observed in 15 pts (48%), grade ≥ 3 was observed in 3 pts (10%). One patient had grade 4 CRS that was later complicated by hemophagocytic syndrome and died on day 21; a second patient had grade 5 CRS in the context of bulky disease and died on day 8. Ten pts (32%) had ICANS and 4 pts had grade 3-4 ICANS. Treatment for CRS / ICANS included tocilizumab (n = 12), siltuximab (n = 4), anakinra (n = 3) and corticosteroids (n = 10). The most common all grade non - hematologic toxicity was fatigue, observed in 19 pts, all grade 1. Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all subjects. Twenty-five (81%) presented disease response and twenty-two pts (71%) achieved complete response (CR). There were no statistically significant differences in the overall and complete response rates between dose levels. After a median follow up of 18 months (range 1 - 32), 5 pts relapsed, and 7 pts have died. Causes of death include progressive disease (n=5), CRS (n=1) and CRS/HLH (n=1). Two-year estimates of PFS and OS for the whole cohort were 67% (95%CI 52-88%) and 75% (95%CI 60-93%)(fig1), respectively. Two-year estimates for patients achieving disease response (CR or PR) were 82% (95%CI 67-99%) and 90% (95%CI 78-100%), respectively. The median duration of response has not been reached (95% CI 74-100). Among pts achieving CR, 94% (95% CI 61-100%) had sustained remission at 12 months. Median time to peak CAR-T expansion, measured by PCR, was 14 days (IQR 14-19), without differences between dose levels, culture duration or fresh vs. cryopreserved infusion. All evaluable subjects had persistent CAR-Ts on PCR measurements done on days 30, 60 and 90. CAR-T cell dose did not have an impact in the time to peak in vivo CAR-T cell expansion or in the rate of CAR-T cell persistence (fig 2). Cytokine measurements have been conducted in 19 pts, with area under the curve (AUC) analyses showing pts with CRS had higher plasma concentrations of multiple cytokines (fig 3). Patients achieving CR had higher plasma concentrations of MIP3B. Conclusions: Second generation antiCD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. On-site manufacture was successful in all pts. This strategy, in combination with fresh product infusion, can make CAR-T cell therapy rapidly available for pts with high-risk r/r B cell lymphoma. Figure 1 Figure 1. Disclosures Caimi: Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria; XaTek: Patents & Royalties: Royalties from patents (wife); Kite Pharmaceuticals: Consultancy; Genentech: Research Funding; ADC Theraputics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Verastem: Consultancy. Ghobadi: Wugen: Consultancy; Atara: Consultancy; Amgen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Schneider: Lentigen Technology: Current Employment. Boughan: Beigene: Speakers Bureau. Metheny: Incyte: Speakers Bureau; Pharmacosmos: Honoraria. Krueger: Lentigen: Current Employment. Kadan: Lentigen: Current Employment. Orentas: Lentigen: Patents & Royalties. Dropulic: Lentigen: Ended employment in the past 24 months, Patents & Royalties. de Lima: Miltenyi Biotec: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: AntiCD19 CAR-T cells with TNFRSF19 transmembrane domain for treatment of relapsed and refractory B cell lymphomas.

Diffuse large B cell lymphoma (DLBCL) is an aggressive but potentially curable malignancy; however, cure is highly dependent on the ability to deliver intensive, anthracycline-based chemoimmunotherapy. Advanced age is an important adverse feature in prognostic models, and nearly one third of cases occur in patients older than 75 years. There is no clear accepted standard of care for older patients with DLBCL due to under-representation of this group in randomized clinical trials. R-miniCHOP, a dose attenuated version of standard R-CHOP, is often chosen based on a prospective, single-arm phase II trial which showed a 2-year progression-free survival (PFS) of 47% (Peyrade, 2011), far lower than observed outcomes in patients younger than 80 yrs (Coiffier, 2002). Precise identification of older DLBCL patients who should be considered for curative versus palliative chemoimmunotherapy is difficult at an individual level; the challenge is to avoid both overtreatment with excessive toxicity and undertreatment with inferior outcomes. The FIL (Fondazione Italiana Linfomi; Italian Lymphoma Foundation) has developed an assessment tool accounting for age, comorbidities, and ability to perform activities of daily living (ADLs) and instrumental activities of living (IADLs). The FIL Tool classifies patients as "fit," "unfit," or "frail." When the FIL tool was prospectively applied to 1163 patients, 55% were fit, 28% were unfit, and 18% were frail. Three-year OS for the whole cohort was 65% and was strongly driven by fitness: 3-year OS was 75% for fit patients, 58% for unfit, and 43% for frail; similar outcomes were seen in a validation cohort (Merli F, 2021). However, treatment was not uniformly directed and was up to the treating physician. Epigenetic deregulation is a feature of DLBCL in older patients, and provides a rationale for targeting methylation. Pre-clinical models show that pre-treatment with hypomethylating agents improves the anti-tumor effect of R-CHOP (Clozel T, 2013). This work has led to early clinical studies of the hypomethylating agent azacitidine with R-CHOP in newly diagnosed DLBCL, with promising early efficacy and acceptable toxicity. The availability of oral azacitidine is an appealing additive approach and is agnostic to cell-of-origin. Based upon the need for improved outcomes in older patients and the promise of azacitidine in this setting, the National Clinical Trials Network (NCTN), led by SWOG, developed S1918, a randomized trial comparing R-miniCHOP to R-miniCHOP + oral azacitidine for older patients (> age 75) with newly diagnosed aggressive B-cell non-Hodgkin lymphomas. This study incorporates the FIL Tool for baseline frailty assessment and a serial comprehensive geriatric assessment (CGA) to evaluate effects of therapy on quality of life and functional status. This is the first randomized study in this population conducted in North America by the NCTN and will enroll a total of 384 eligible patients after a safety run-in phase. All patients will receive pre-phase therapy with vincristine 1mg x 1 and prednisone 60mg/m2 x 7 days (capped at 100mg/day); pre-phase therapy has been shown to improve performance status and decrease early treatment-related mortality (Owens CN, 2015). The primary objective of the phase II component is to determine if oral azacitidine + R-miniCHOP regimen should be tested further (Phase III) against R-miniCHOP alone based on estimates of 1-year progression-free survival (PFS). The primary objective of the phase III component is to compare the OS at 2 years between oral azacitidine + R-miniCHOP and R-miniCHOP alone. Key correlative tests will include circulating tumor DNA (ctDNA) assays at pre-specified timepoints to explore if ctDNA quantity and methylation patterns correlate with response. S1918 has the potential to impact future trial design and to change the standard of care for patients 75 years and older with aggressive lymphoma in a number of ways given its randomized design, incorporation of geriatric assessments, and utilization of ctDNA correlatives. The prospective assessment of frailty, serial comprehensive geriatric assessments, and introduction of epigenetic modulation will hopefully improve outcomes for this population with significant need. Trial registration: ClinicalTrial.gov Identifier NCT04799275 Funding: NIH/NCI/NCTN grants U10CA180888, U10CA180819, U10CA180820, and U10CA180821 Figure 1 Figure 1. Disclosures Brem: Morphosys/Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pharmacyclics/Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; TG Therapeutics: Consultancy; SeaGen: Speakers Bureau; KiTE Pharma: Membership on an entity's Board of Directors or advisory committees. Caimi: Seattle Genetics: Consultancy; Verastem: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Amgen Therapeutics.: Consultancy; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy; ADC Theraputics: Consultancy, Research Funding; TG Therapeutics: Honoraria. Cerchietti: Bristol Myers Squibb: Research Funding; Celgene: Research Funding. Alizadeh: Foresight Diagnostics: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Forty Seven: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; CAPP Medical: Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Cibermed: Consultancy, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company; Roche: Consultancy, Honoraria; Janssen Oncology: Honoraria; Celgene: Consultancy, Research Funding; Gilead: Consultancy; Bristol Myers Squibb: Research Funding. Olin: Amgen: Honoraria; Cellectis: Research Funding; Pfizer: Research Funding; Daiichi Sankyo: Research Funding; Genentech: Research Funding; Abbvie: Honoraria; Actinium: Honoraria; Astellas: Honoraria, Research Funding. Friedberg: Novartis: Other: DSMC ; Bayer: Other: DSMC ; Acerta: Other: DSMC . Smith: Celgene, Genetech, AbbVie: Consultancy; Alexion, AstraZeneca Rare Disease: Other: Study investigator.

Background: Chimeric antigen receptor T cell (CAR-T) therapy is known to produce durable remissions in the treatment of CD19 + relapsed/refractory B cell malignancies. Nonetheless, many patients receiving CAR-T cells will fail to respond for unknown reasons. Here, we employed single cell RNA sequencing and protein surface marker profiling of serial CAR-T cell samples from patients with non-Hodgkin's lymphoma (NHL) to reveal CAR-T cell evolution, identify biomarkers of response, and test for evidence of exhaustion in CAR-T cells of poor responders. Methods: To describe the evolution of CAR-T cells after infusion into NHL patients and to identify mechanisms and biomarkers of response, our study examined manufactured CAR-T cell products and FACS isolated CAR-T cells from post-infusion blood samples from patients treated for CD19 + relapsed/refractory NHL. Utilizing scRNA sequencing and flow cytometry, we investigated time points after infusion that are known from previous studies to be associated with peak expansion (day 14) and contraction (day 30) and represent key changes in CAR-T cell activity. Altogether, our datasets include 14 manufactured CAR-T cell products, 13 samples from day 14, and 12 samples from day 30. This sampling represents 10 patients with favorable response (complete or partial remission (CR; PR)) and 4 patients with poor response (stable or progressive disease (SD; PD)). To isolate CAR-T cells for scRNA sequencing, viable CD3 +CAR + cells were sorted from cryopreserved CAR-T cell products or PBMCs. Next, libraries were generated with the 10x Genomics Chromium single cell 3' platform with feature barcoding technology to allow simultaneous and paired quantification of transcriptional and cell surface protein expression in individual CAR-T cells. The libraries were sequenced and the data stringently filtered. Batch effect removal was applied to remove differences due to sample preparation or sequencing. Results: Post-filtering, our dataset contained 94,000 cells with an average of 3,917 cells per sample, 8,518 reads per cell, and 2263 unique detectable genes per cell. After dimension reduction and clustering, eleven high-frequency clusters were identified with cluster patterns corresponding with time point, cell cycle phase, cell type, or patient (Figure 1). At the transcriptional level, post-infusion CD8 CAR-T cells displayed significant upregulation of transcription factors (PRDM1, EOMES) and cytotoxic effector molecules (GZMB, PRF1, GZMK, CCL5) associated with differentiation into cytotoxic effector cells as well as transcription factors associated with exhaustion (TOX, TOX2, NR4A2, NR4A3) (p.adj < 0.05) (Figure 2). These data were corroborated by enrichment of effector and exhaustion gene set signatures after infusion as well as global increases in the protein expression of activation and exhaustion markers CD45RO, CD69, CD57, PD1 (CD279), and TIGIT. Contrasts of CAR-T cells between response groups displayed enrichment of an exhaustion profile in CD8 CAR-T cells of poor responders characterized by significant upregulation of the transcription factors FOS, JUNB, JUND, FOSB, JUN, NR4A2, NFKBIA, and PRDM1 and other markers of exhaustion at the RNA level (p.adj < 0.0001). At the protein level, the frequency of TIGIT expression was 20% greater on average in the CD8 CAR-T cells of poor responders compared to favorable responders (Figure 3). CD8 CAR-T cells expressing TIGIT compared to TIGIT negative CD8 CAR-T cells were enriched in an exhaustion transcriptional profile by gene set enrichment analysis and expressed consistently higher levels of exhaustion markers (PD1, CTLA4, LAG3, TIM3) by flow cytometry, regardless of response group or time point (Figure 4). Conclusions: At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a non-proliferative, highly-differentiated, and exhausted state that is enriched in CAR-T cells of patients with poor response. Furthermore, we identified the checkpoint receptor TIGIT as a novel prognostic biomarker and potential driver of CAR-T cell exhaustion. Figure 1 Figure 1. Disclosures Dropulic: Lentigen: Ended employment in the past 24 months, Patents & Royalties. Caimi: Kite Pharmaceuticals: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Seattle Genetics: Consultancy; Genentech: Research Funding; ADC Theraputics: Consultancy, Research Funding; Verastem: Consultancy; Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria. de Lima: Miltenyi Biotec: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees.

Introduction: Approximately one-third of patients (pts.) with diffuse large B-cell lymphoma (DLBCL) develop relapsed or refractory (R/R) disease after frontline rituximab-(R) based chemo immunotherapy. Variability in expression of the B-cell surface antigen CD20, and possibly CD19, is thought to be an important mechanism of treatment failure, but there is vast heterogeneity in the reported incidence, with most reports utilizing Immunohistochemical (IHC) staining which is non-quantitative in nature. New therapeutics including CAR T-cells, antibodies (Ab) (tafasitimab) and Ab-drug conjugates (loncastuximab teserine) target CD19, while several bispecific engager Abs. in development target CD20. Understanding the evolution of target antigen expression in R/R DLBCL can provide information regarding selection and sequencing of therapies. We sought to quantitate the change in surface expression of CD19 and CD20 in R/R DLBCL after R-based chemo immunotherapy relative to pretreatment levels, to provide insight into the relative stability of these targets. Methods: Pts. diagnosed with DLBCL after January 2014, who were R/R to frontline R-based chemo immunotherapy (R-CHOP or R-EPOCH) were retrospectively identified. Pt. and disease related clinical variables at diagnosis (Dx) and first R/R were recorded. Data from IHC were obtained, as were archival flow cytometry assays performed on tumor biopsies at Dx and in the R/R setting. Using FCS Express® software, neoplastic cells were gated, and fluorescence intensity (FI) of CD19 and CD20 expression were reported (using fluorochromes APC-A for CD19 and PECy7 for CD20, respectively). Multivariate linear mixed model was used to compare median and geometric mean FI of CD19 and CD20 between Dx and R/R, while adjusting for other clinical variables. Results: A total of 51 flow cytometry assays (26 Dx and 25 R/R) were analyzed for 33 pts. Median age at Dx was 64 (range, 41-76) yrs., 24 pts. (73%) were male and 29 (88%) had IPI ≥ 2. 11 (33%) pts. had a prior indolent lymphoma. Cell of origin at Dx was GCB in 16 (49%) and non-GCB in 12 (36%) pts., while two had a double-hit rearrangement at diagnosis. Treatment was R-CHOP in 27 cases (82%) and R-EPOCH in 6 (18%). Median time to R/R was 10.4 months. There was a significant reduction in median FI of CD20 from Dx [median: 40,610 (range: 167 - 259,962)] to R/R [median: 11,596 (range: 63 - 79,592)], representing a mean reduction of 63% at R/R relative to Dx (P= 0.01; 95% CI: 20-73%). Similar change was observed in geometric mean FI of CD20, which was reduced 65% at R/R relative to Dx (P< 0.01; 95%CI: 31-82%). In comparison, on IHC, CD20 was reported to be negative at R/R relative to Dx in only one of 16 pts. for whom IHC results were available. Median and geometric mean FI of CD19 at R/R were 38% and 20% lower compared to Dx, respectively, but these differences were not statistically significant (P= 0.08 and 0.39, respectively) (Table). When examining the relative change in FI at R/R in individual cases, compared to the mean FI of all Dx cases, we observed that 21 out 25 R/R cases (84%) had reduction of CD20 whereas only 14/25 (56%) had reduction of CD19. Interestingly, 7/25 (28%) R/R cases had an increase in CD19 expression by >80% (Figure).When adjusting for clinical variables such as age, sex, presence of B symptoms, bone marrow (BM) involvement, PS and prior indolent lymphoma, the change in CD20 median FI from Dx to R/R maintained statistical significance (p=0.01). Reduction in CD20 geometric mean FI from Dx to R/R was significantly associated with age >60 years (p=0.04), BM involvement (p 1 site of extra nodal involvement (p= 0.03) at Dx. Conclusions: Quantitative assessment by flow cytometry revealed a significant decline in expression of CD20 at R/R compared to Dx in the majority of patients with DLBCL treated with R-based chemo immunotherapy. CD19 expression was unchanged in most R/R cases but was found to be dramatically upregulated in a subset of R/R cases. Given the role of CD19 mediated pathways in B-cell NHL and its association with PI3K pro-survival signaling, these data merit further exploration as a potential mechanism of treatment resistance. These findings also highlight the importance for repeat tissue biopsy at the time of suspected R/R DLBCL, with focus on therapeutic target expression, as it may influence treatment decisions or enrollment in clinical trials exploring efficacy of newer agents. Figure 1 Figure 1. Disclosures Caimi: TG Therapeutics: Honoraria; Seattle Genetics: Consultancy; Amgen Therapeutics.: Consultancy; XaTek: Patents & Royalties: Royalties from patents (wife); Verastem: Consultancy; Genentech: Research Funding; Kite Pharmaceuticals: Consultancy; ADC Theraputics: Consultancy, Research Funding. Hill: Celgene (BMS): Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Other: Travel Support, Research Funding; Karyopharm: Consultancy, Honoraria, Research Funding; AstraZenica: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Gentenech: Consultancy, Honoraria, Research Funding; Incyte/Morphysis: Consultancy, Honoraria, Research Funding; Beigene: Consultancy, Honoraria, Research Funding.

In people with cancer, pain often occurs from the malignancy, from procedures done to diagnose, stage, and treat the malignancy, and from the toxicities of therapy used in treating the cancer. Of people with cancer, 75% complain of some sort of pain. Determining whether the pain is from tissue damage or nerve structures will guide therapy. Assessment of the severity of the pain by location, oncological type, as well as psychosocial and environmental factors are necessary to understand and treat the pain that accompanies cancer. Medical interventions include non-opioid analgesics, opioids, and multiple different combinations of medications. Adjuvant medication like anticonvulsants and steroids are being used frequently to help people feel more comfortable.