Villasboas, José C.

Background CD8+ T-lymphocyte subsets defined by killer lectin-like receptor G1 (KLRG1) and CD127 expression have been reported to have an important role in infection, but their role in the setting of lymphoid malignancies, specifically follicular lymphoma (FL), has not been studied. Methods To characterize the phenotype of KLRG1/CD127-defined CD8+ subsets, surface and intracellular markers were measured by flow cytometry and Cytometry by time of flight (CyTOF), and the transcriptional profile of these cells was determined by CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). The functional capacity of each subset was determined, as was their impact on overall survival (OS) and event-free survival (EFS) of patients with FL. Results We found that intratumoral CD8+ cells in FL are skewed toward effector cell subsets, particularly KLRG+CD127- and KLRG1-CD127- cells over memory cell subsets, such as KLRG1-CD127+ and KLRG1+CD127+ cells. While effector subsets exhibited increased capacity to produce cytokines/granules when compared with memory subsets, their proliferative capacity and viability were found to be substantially inferior. Clinically, a skewed distribution of intratumoral CD8+ T cells favoring effector subtypes was associated with an inferior outcome in patients with FL. Increased numbers of CD127+KLRG1- and CD127+KLRG1+ were significantly associated with a favorable OS and EFS, while CD127-KLRG1- correlated with a poor EFS and OS in patients with FL. Furthermore, we demonstrated that interleukin (IL)-15 promotes CD127-KLRG1+ cell development in the presence of dendritic cells via a phosphoinositide 3-kinase (PI3K)-dependent mechanism, and treatment of CD8+ T cells with a PI3K inhibitor downregulated the transcription factors responsible for CD127-KLRG1+ differentiation. Conclusions Taken together, these results reveal not only a biological and prognostic role for KLRG1/CD127-defined CD8+ subsets in FL but also a potential role for PI3K inhibitors to manipulate the differentiation of CD8+ T cells, thereby promoting a more effective antitumor immune response.

BACKGROUND Cytometry by time‐of‐flight (CyTOF) or mass cytometry is a form of flow cytometry. CyTOF uses antibodies bound to metal isotopes as tags instead of fluorochromes normally used in standard flow cytometry. These nonradioactive isotopes of heavy metals allow mass cytometry to analyze up to 50 different parameters without significant signal overlap. Due to the accuracy of mass cytometry, researches are able to investigate multiple cell markers at once, which is not possible with standard fluorescent flow cytometry (FFC). CyTOF works by feeding a cell sample tagged with conjugated antibodies into a nebulizer where cells are aerosolized. The aerosolized samples pass through an argon plasma torch and become an ionized atom cloud. This cloud passes through a quadrupole where low mass ions are filtered out resulting in a cloud enriched for the heavy metal reporter tags. The tags enter the time‐of‐flight chamber and further separated by mass as they reach the detector AIMS The main aim of this project was to optimize the stimulation conditions that are best suited for use with a CyTOF T cell functional panel. This would provide a method for decreased cell loss in sample preparation. METHODS T‐cell‐enriched samples underwent stimulation using 4 different condition: 4‐hour stimulation of PMA/Ionomycin, 1:1 ratio of Dynabeads at 24h, 48h, and 72h. Brefeldin A was placed in every well containing cells to act as transport inhibitor. FFC was used to test cell recovery and cell death between stimulation techniques and stimulation strength was observed using CyTOF. RESULTS There was no significant difference in 4h unstimulated cells and 24h unstimulated cells in both cell recovery and cell viability. However, comparing the PMA/Ionomycin unstimulated cells to the 4h stimulated cells did produce a significant decrease in cell recovery and cell death. Because this technique is a non‐physiological stimulation, this result was expected. The use of FFC also allowed us to see the significant loss of cells in recovery and death after 24h stimulation with the 48h and 72h Dynabead assays. The “sledge hammer” effect is clearly visible with the PMA/Ionomycin with 2.73% of IL‐4 cells being stimulated compared to only 0.28% in the 24h Dynabead sample. This trend of PMA/Ionomycin producing a strong stimulation signal was a trend. CONCLUSIONS We determined that T cell culture with Brefeldin A beyond 24 h significantly decreases cell viability and recovery. In observing the stimulation for Dynabeads at 24h, the cells are not directly affected while PMA/Ionomycin does cause a significant loss in cell viability and total cells recovered. PMA/Ionomycin is capable of stimulating the production of all measurable cytokines, even rare populations such as IL‐10‐ or IL‐6‐producing T cells. Dynabeads resemble a more physiological stimulation condition and is able to activate all T cell subsets except IL‐6‐producing T cells. Support or Funding Information The Berner Charitable and Scholarship Foundation, Funded by the Immune Monitoring Core and The Division of Hematology This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

e19556 Background: CD19 chimeric antigen receptor T cell therapy possesses unique side effects including cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). Age is a major risk factor for ICANS. However, whether ICANS in older patients is different compared to younger patients is unknown. Herein, we report clinical course, outcomes and risk factors for ICANS in older patients with large B cell lymphoma (LBCL) treated with axicabtagene ciloleucel (axi-cel). Methods: We comprehensively reviewed detailed clinical courses of ICANS in 78 adult patients with LBCL treated with axi-cel between June 2016 and October 2020. Incidence, manifestation, risk factors, treatment, and outcomes of ICANS were compared between patients age ≥60 (n=32) and <60 (n=46) years old. Results: Baseline characteristics were comparable between older and younger patients except higher proportion of high international prognostic index and underlying cerebral microvascular disease in older patients. ICANS was observed in 16 patients in the older and 24 patients in the younger age group, with a 30-day incidence of 52% and 50%, respectively. Median time to CRS and ICANS were similar between 2 age groups. The most common initial neurological findings included aphasia, dysgraphia and encephalopathy in both age groups. Table summarizes the characteristics, clinical course and interventions of ICANS in older and younger patients. In Cox regression model, the presence of CRS was the only factor associated with ICANS in both age groups. Age, history of central nervous system involvement and cerebral microvascular disease were not associated with ICANS. Importantly, all patients had complete resolution of ICANS. No elderly patients in our cohort experienced seizure as a manifestation of ICANS. Conclusions: In our study, older age was not a risk factor for ICANS. CRS was the only factor associated with ICANS in both younger and older patients. Incidence, clinical course and neurological outcomes of ICANS in older patients treated with axi-cel were comparable to younger patients. [Table: see text]

e19554 Background: Obesity is associated with a pro-inflammatory state and immune dysregulation. Retrospective studies indicate that obesity could affect toxicities and outcomes after immunotherapies including checkpoint inhibitors and allogeneic stem cell transplant. Currently, there are no data specifically on outcomes for obese patients who receive chimeric antigen receptor T (CAR-T) cells. We described the clinical outcome in obese patients with large B cell lymphoma (LBCL) who received axicabtagene ciloleucel (axi-cel). Methods: We analyzed the effect of body weight (BW) and body mass index (BMI) on toxicities and outcomes of 78 adults with LBCL who received axi-cel between June 2016 and October 2020 at Mayo Clinic. Obesity was defined as having BMI of 30 or higher. Results: Of 78 patients, 22 (28%) and 19 (24%) were classified as overweight (BMI 25-29.99 kg/m2) and obese (BMI ≥30 kg/m2), respectively. Baseline characteristics were not statistically significantly different between non-obese and obese patients. The median delivered dose of fludarabine was similar between non-obese and obese patients (89 [0-105] vs 88 [56-94] mg/m2, P=0.32) whereas the median delivered dose of cyclophosphamide was lower in non-obese patients (1503 [1077-1525] vs 1512 [1021-1660] mg/m2, P=0.01). The 30-days cumulative incidence of CRS and ICANS were similar between non-obese and obese patients. BW and BMI were not associated with CRS or ICANS. The overall response rate was 66% (CR 47%) and 68% (CR 53%) in non-obese and obese group, respectively ( P=0.83). The 1-year event free survival (EFS) and overall survival (OS) was 34.6% and 64.5%, neither were different between non-obese and obese patients (EFS 35.8% vs. 30.7%, P=0.60; OS 59.4% vs. 83.9%, P=0.18). The 1-year cumulative incidence of relapse and non-relapse mortality was comparable (60.8% vs. 69.0%, P=0.40 and 3.4% vs. 0%, P=0.42). In the Cox proportional hazards model, higher dose of fludarabine, but not cyclophosphamide, was associated with better EFS and OS; however, neither obesity nor BW were associated with toxicities and outcomes. Conclusions: In our study, Obesity was not associated with risk of toxicities or adverse survival outcomes. The effect of obesity on the pattern of LD chemotherapy dosing including toxicities and outcomes after CAR-T warrants further exploration. [Table: see text]

e19547 Background: Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL, LT) is a rare, aggressive lymphoma characterized by skin involvement predominantly in the lower extremities, and is associated with an inferior prognosis compared to other primary cutaneous B-cell lymphomas. Immunochemotherapy with or without involved-field radiation therapy (IFRT) is considered standard, front-line therapy. Interestingly, over-expression of PD-L1/PD-L2 has been shown in a high proportion of PCDLBCL, LT cases, but efficacy of immune checkpoint inhibitors (ICI) in relapsed/refractory, PCDLBCL, LT has not been thoroughly studied. Therefore, we describe the outcomes of 1) immunochemotherapy with and without IFRT as front-line treatment of PCDLBCL, LT, and 2) ICIs in the relapsed/refractory setting. Methods: We conducted a retrospective cohort study of patients diagnosed with PCDLBCL, LT seen at Mayo Clinic from January 1, 2000 to December 31, 2020. Using the Kaplan-Meier method, we calculated progression-free survival (PFS), duration of response (DOR), and overall survival (OS) in patients who received front-line R-CHOP with and without IFRT, and salvage ICI therapy for relapsed/refractory disease. Results: A total of 28 patients with PCDLBCL, LT were identified. The median age at diagnosis was 71.6 years (range 48.0-91.7), 50% (N = 14) were male, and 78.6% (N = 22) had disease involvement of the lower extremities at diagnosis. For front-line treatment, 31.2% (N = 9) received R-CHOP with IFRT, and 31.2% (N = 9) received R-CHOP without IFRT. The median PFS in patients treated with R-CHOP plus IFRT was 41.8 months [95% CI: 30.2-69.6] compared to 13.7 months [95% CI: 10.7-27.7; p= 0.01] in those treated with R-CHOP without IFRT. The median OS in patients treated with R-CHOP plus IFRT was 74.7 months [95% CI: 53.0-108.6] compared to 38.2 months [95% CI: 26.0-80.4; p= 0.14] in those treated with R-CHOP without IFRT. Patient and disease characteristics were similar among these two groups. ICIs were used in 17.9% (N = 5) of patients with relapsed/refractory, PCDLBCL, LT, and these patients had received a median of three (range 2-10) prior systemic therapies. The overall response rate was 60% as three patients treated with ICIs achieved a complete response, and the other two patients showed no clinical response. The median DOR from ICIs was 23.0 months [95% CI: 3.6-26.0]. The median PFS from ICI therapy was 10.2 months [95% CI: 3.6 – not reached]. Only one of the five patients was noted to have a mild side effect from ICI treatment (elevated alkaline phosphatase, grade 1). Conclusions: R-CHOP with IFRT was associated with a longer median PFS compared to R-CHOP without IFRT as front-line therapy for PCDLBCL, LT. Furthermore, ICIs may have a role in treating relapsed/refractory disease as reasonable activity in heavily pre-treated patients and a favorable safety profile were observed in this study. Further studies would be of benefit to confirm our findings.

e19501 Background: The current standard of care for patients with relapsed or refractory (RR) diffuse large B-cell lymphoma (DLBCL) following frontline immunochemotherapy (IC) is salvage therapy, followed by high-dose chemotherapy and autologous stem cell transplant (ASCT) rescue in patients responding to salvage therapy. Time to first relapse (or refractory status) and response to salvage therapy in patients with RR DLBCL may reflect the chemosensitivity of the underlying disease. The aim of this study is to determine whether these factors impact post-ASCT outcomes. Methods: Patients with DLBCL that relapsed after R-CHOP or R-CHOP-like frontline therapy who underwent salvage therapy and ASCT at Mayo Clinic or University of Iowa between 07/2000 and 4/2020 were identified from institutional lymphoma and transplant databases. Clinical characteristics, treatment information, and outcome data were abstracted. Progression-free survival (PFS) and overall survival (OS) from the time of ASCT were analyzed using Kaplan-Meier method and Cox proportional hazards models. Results: A total of 437 patients with RR DLBCL who underwent salvage therapy and ASCT were identified. 280 (64%) were male. Median time from initial diagnosis to 1st relapse/salvage was 1.0 years (range 0.1-16.4). A median of 1 line (range 1-3) of salvage therapy was required. Response prior to ASCT was complete response (CR) in 211 (48%), partial response in 199 (46%), stable disease in 24 (5%), and unknown in 3 (1%) patients. Median age at ASCT was 61 years (range 19-78), and median follow up after ASCT was 8.0 years (95% CI 7.2-8.7). Median PFS and OS was 2.7 (95% CI 1.5-4.3) and 5.4 (4.2-7.4) years, respectively. Time to 1st relapse/salvage (≤1 vs 1-2 vs > 2 years) was not associated with PFS (median 0.8 vs 2.4 vs 4.9 years, p = 0.170) but was associated with OS (2.4 vs 7.4 vs 6.8 years, p = 0.013). Patients who required > 1 line of salvage therapy had significantly inferior PFS (median 0.3 vs 4.5 years, p < 0.001) and OS (0.9 vs 7.4 years, p < 0.001). In addition, patients who failed to achieve a CR prior to ASCT had significantly worse PFS (median 0.8 vs 5.3 years, p < 0.001) and OS (2.7 vs 9.2 years, p < 0.001). In multivariate Cox regression models adjusted for age and sex, time to 1st relapse/salvage was not associated with PFS (p = 0.313) or OS (p = 0.081); however, lines of salvage and response prior to ASCT remain significantly prognostic for PFS ( > 1 line of salvage: HR 2.14, 95% CI 1.59-2.87, p < 0.001; non-CR: HR 1.61, 95% CI 1.26-2.05, p < 0.001) and OS ( > 1 line of salvage: HR 2.25, 95% CI 1.66-3.05, p < 0.001; non-CR: HR 1.63, 95% CI 1.26-2.12, p < 0.001). Conclusions: In patients with RR DLBCL following frontline IC, requiring more than 1 line of salvage therapy and failure to achieve CR are strong independent risk factors for poor PFS and OS after ASCT. Novel therapies such as CAR-T cell therapy should be studied in this population.