Peak absolute lymphocyte count after <scp>CAR‐T</scp> infusion predicts clinical response in aggressive lymphoma
Authors: Radhika Bansal, Mattia Novo, Abdullah S. Al Saleh, Anatilde Gonzalez Guerrico, Henan Zhang, Zuoyi Shao, Elham Babadi, Kodi E. Martinez, Gabrielle A. McCoy, Matthew A. Hathcock, Arushi Khurana, Nabila Nora Bennani, Jonas Paludo, Yucai Wang, Stephen M. Ansell, Patrick B. Johnston, Jose C. Villasboas, Yi Lin
Journal: American Journal of Hematology (2022)

Chimeric antigen receptor T (CAR-T) cell therapy has been a breakthrough in personalized cancer therapy, with US Food and Drug Administration approved CD19 CAR-T products available now for the treatment of relapsed/refractory aggressive B-cell non-Hodgkin's lymphoma (r/r NHL) for close to 4 years.1 The rate of durable response after CAR-T has been reported to be 30%–40% across various registration studies.2, 3 CAR-T expansion has been consistently correlated with clinical response.2-4 The median time to peak CAR-T expansion can vary based on the CAR construct, but generally occurs in the first 2 weeks of infusion.2-4 CAR T-cell expansion kinetics is monitored by lab-based assays like polymerase chain reaction or flow cytometry for research purposes. Currently, there is no clinically available test to assess CAR-T expansion in SOC practice. We hypothesize that since patients are lympho-depleted from chemotherapy prior to CAR-T infusion, the lymphocyte expansion in the first 2 weeks post-CAR-T infusion is due primarily to CAR-T expansion. Thus, peak absolute lymphocyte count (ALCpeak), readily accessible by clinical complete blood count (CBC) with differential, can be a surrogate for CAR-T expansion. In this study, we examined the ALC levels in the first 2 weeks after CAR-T infusion and the correlation between CAR-T levels and clinical outcomes for NHL patients who received CAR-T at our institution. We conducted a retrospective review of electronic medical records of patients who received anti CD19 CAR-T for treatment of aggressive, r/r NHL between 6/2016 and 1/2021 at Mayo Clinic, Rochester. ALC was collected daily from serial complete blood cell counts with differentials. The maximum ALC value in the first 15 days of CAR-T infusion was identified as ALCpeak. CAR-T outcomes were obtained from the prospectively collected and validated immune effector cell program clinical database. A durable response was defined as complete remission (CR) at month 6 and beyond. Sixteen patients consented to participate in the biomarker study. Research blood samples were analyzed by flow cytometry (Cytoflex and Cytoflex LX) for CAR-T quantification. CAR-T quantification was done at 2 h post infusion, day 1, day 7, and day 14 post infusion. The highest value on day 7 or day 14 was taken as the peak CART number, which is within the range of published axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) expansion time frame. Immunophenotyping was performed for CAR-T cells using canonical antibodies for T cell lineage and FMC63-PE antibody for CAR expression according to the manufacturer's recommendations. Flow data were analyzed using Kaluza software, version 2.2. The receiver operating characteristic (ROC) curve was used to determine the best cut-off of ALCpeak in predicting CR as the best response. Event-free survival (EFS) was defined as the time from CAR-T infusion to the time of PD, next therapy, or death. Overall survival (OS) was measured from the date of CAR-T infusion to the date of death or last follow-up. EFS and OS were analyzed using Kaplan–Meier analysis and statistical significance was assessed using the two-tailed log-rank test. The Cox proportional hazards model was used to evaluate the association of ALCpeak and other prognostic factors with EFS and OS. Multivariate analysis was done using Cox regression models and tested all variables with a p ≤ .05 in the univariate analysis. χ2-tests were used to determine relationships between categorical variables. The Wilcoxon-rank test was used to determine associations between continuous variables and categories, and Spearman correlation coefficients were used to evaluate associations for continuous variables. Among the 87 patients who received CAR-T for NHL, 86 (99%) of the patients received axi-cel and 1 (1%) received tisa-cel. (Table S1) The median age at the time of CAR-T evaluation for this cohort was 58 (range, 26–76) years. All patients received lymphodepletion chemotherapy with fludarabine and cyclophosphamide from day −5 to day −3. The median and range of ALC on the day of CAR-T infusion were 0.02 × 109/L (IQR 0.02–0.03 × 109/L). The median ALCpeak for all patients was 0.44 × 109/L (range, 0–2.55 × 109/L). The median time to ALCpeak was 10 ± 3 days (Figure S1A). The incidence of CRS was 86%, 6% for grade 3 or higher. The incidence for ICANS was 53%, and 17% for grade 3 or higher (Table S2). The CR rate was 52% and the PR rate was 17%. Durable response, defined as CR through month 6, was seen in 31% of patients. With a median follow-up was 20 months (range: 1.14–59.3), the median EFS and OS were 3.2 (95% CI: 2.9–4.7) months and 14.9 (95% CI: 11.9–31.4) months respectively. As we hypothesized that initial ALC expansion in the first 15 days would be due to CAR-T expansion rather than hematopoietic recovery from chemotherapy, we examined the correlation of ALCpeak with CAR-T levels. Increased ALCpeak correlated with increased CAR+ T cells in blood by flow (n = 16, R = 0.63, p = .0008, Figure 1A). Patients who achieved CR as the best response (Figure S1B) and those who had durable CR (Figure 1B) had a higher ALCpeak versus those who did not. Using ROC analysis, ALCpeak level of 0.67 was identified as the cut point for association with complete remission as the best response (AUC = 0.68, p = .0004). The only statistically significant difference between high ALCpeak (≥0.67 × 109/L, N = 35) versus low ALCpeak (<0.67 × 109/L, N = 52) was gender (Table S1). There was no difference in the incidence, grade ≥3, or duration of CRS and ICANS between the two groups. As steroids can impact blood counts, we examined the pattern of steroid use post-CAR-T infusion for the management of CRS and ICANS. The median and range to the start of steroid use were 5 days (range, 1–12). The median duration of use of steroids is 4 days (range, 1–17). Out of the 30 patients who received steroids, 25 (83%) patients received steroids prior to reaching peak ALC. The ALCpeak of these 25 patients was 0.40 ± 0.56 cells/uL, compared to patients who never received any steroids (n = 57, 0.44 ± 0.52 cells/uL, p = 0.9) As expected, patients who received a higher cumulative dose of steroid had lower ALCpeak. Patients with high ALCpeak after CAR-T infusion had longer EFS [6.4 months vs. 2.7 months, p = .004, Figure 1C] and OS [31 months vs. 12 months, p = .03, Figure 1D] compared with patients with low ALCpeak. In the univariate analysis by the Cox model, high ALCpeak and IPI score ≥3 were associated with prolonged EFS. High ALCpeak and age ≥ 60 years also correlated with OS on univariate analysis. However, in the multivariate analysis by the Cox model, ALCpeak was the only variable that remained an independent predictor for EFS (HR = 0.47, 95% CI, 0.28–0.8, p = .005) and OS (HR = 0.52, 95% CI, 0.27–0.97, p = .04). CAR-T cell expansion, which is associated with clinical response, is currently measured for research purposes only. ALC is clinically accessible in the lab test complete blood count with differential. The importance of early lymphocyte recovery at day +15 has been well characterized in autologous stem cell transplant (ASCT) as a significant predictor for tumor control and survival in hematologic malignancies including NHL and HL.5 Gordan et al. demonstrated that ALC ≥0.667 × 109/L at day +15 after ASCT independently predicts better EFS in NHL and HL.5 In CAR-T therapy, the mechanism of lymphocyte expansion in the first 15 days is different from ASCT and is due to the expansion of infused CAR-T product rather than from engraftment of stem cells and subsequent hematopoiesis. Nevertheless, our study demonstrates that differential counts, which are routinely available in the clinic, can be used as a surrogate for CAR-T expansion and correlate with clinical response. In our cohort of patients, ALCpeak in the first 15 days correlated with CAR-T cell levels. CAR-T expansion can differ depending on the CAR-T19 products, the patients' immune cell fitness, and the type of disease. Different CAR-T products may have different cellular kinetics due to patient-specific factors: T cell composition and fitness, preinfusion tumor burden, use of lymphodepleting regimens, steroid use and an anti-CAR immune response. ALC expansion kinetics post-CAR-T infusion could be a useful surrogate for CAR-T levels given the general timing of CAR-T expansion and hematopoietic recovery from lymphodepletion chemotherapy. Our study also supports that ALCpeak was predictive of the treatment response. ZUMA-1 study primary analysis reported that higher CAR-T cell peak concentrations in peripheral blood were associated with durable response. In our study, ALCpeak was higher in patients who achieved CR and durable CR for 6 months or more. ALCpeak was an independent predictor for prolonged EFS and OS by multivariate analysis. Hence, ALCpeak as a surrogate for CAR-T expansion can predict clinal response and risk of progression. The ALCpeak cut-off level in our report may be predictive of clinical outcomes specifically with axi-cel, given the relative fewer use of other CAR-T products. Larger, prospective studies can validate our findings with axi-cel and determine predictive ALCpeak timing and values with other CAR-T therapy. This can be a useful, readily available clinical test for clinicians at the bedside. In conclusion, CBC with differential is an easily accessible clinical lab that can be used in the standard of care practice as a surrogate to estimate relative CAR-T expansion. It can help predict clinical outcomes and survival in lymphoma patients receiving CAR-T. ALCpeak threshold needs to be validated with prospective, multi-center studies and will likely vary for CAR-T with different expansion kinetics. This study was supported in part by the Mayo Clinic Center for Individualized Medicine, Bernard E. and Edith B. Waterman, Henry J. Predolin Foundation, and other generous benefactors of Mayo Clinic. We thank the patients and their families for their dedication and participation in the research. JP: Karyopharm, WY: Lncyte, lnnoCare, LOXO Oncology, Novartis, Genentech, MorphoSys, Eli Lilly, TG Therapeutics, SA: Bristol Myers Squibb, ADC Therapeutics, Seattle Genetics, Regeneron, Affimed, Al Therapeutics, Pfizer, Trillium and Takeda, YL: Kite, a Gilead Company, Janssen, Novartis, Celgene, Bluebird Bio, Juno, Legend, Sorrento, Gamida Cell, Vineti. The remaining authors declare no competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript. Yi Lin designed the study and contributed to statistical analysis, Anatilde Gonzalez Guerrico, Henan Zhang, Zuoyi Shao, Elham Babadi, Kodi E Martinez, Gabrielle A. McCoy performed laboratory research and contributed to data collection, Radhika Bansal collected and analyzed the data, and wrote the initial manuscript draft, Matthew A. Hathcock, Arushi Khurana, Nabila Nora Bennani, Jonas Paludo, Yucai Wang, Stephen M. Ansell, Jonas Paludo, Jose C Villasboas, Yi Lin critically reviewed the manuscript drafts, approved the final version, and made the decision to submit the manuscript for publication. The data that supports this study is available on request from the corresponding author upon reasonable request. The data that supports this study is available on request from the corresponding author upon reasonable request. Data S1 Supporting information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.