Biomarkers for cytopenias following CAR T-cell treatment in relapsed/refractory (RR) multiple myeloma (MM) are not completely defined. We prospectively analysed 275 sequential peripheral blood (PB) ...samples from 58 RRMM patients treated with BCMA-targeted CAR T cells, and then divided them into three groups: (i) baseline (before leukapheresis), (ii) ≤day+30, and (iii) >day+30 after CAR T-cell therapy. We evaluated laboratory data and performed flow cytometry to determine the (CAR) T-cell subsets. Baseline hyperferritinaemia was a risk factor for long-lasting grade ≥3 anaemia (r = 0.47, p < 0.001) and thrombocytopenia (r = 0.44, p = 0.002) after CAR T-cell therapy. Low baseline haemoglobin (Hb) and PLT were associated with long-lasting grade ≥3 anaemia (r = -0.56, p < 0.001) and thrombocytopenia (r = -0.44, p = 0.002) respectively. We observed dynamics of CAR-negative T-cell subsets following CAR T-cell infusion. In the late phase after CAR T-cell therapy (>day+30), CD4Tn frequency correlated with anaemia (r = 0.41, p = 0.0014) and lymphocytopenia was related to frequencies of CD8
T cells (r = 0.72, p < 0.001) and CD8Teff (r = 0.64, p < 0.001). CD4Tcm frequency was correlated with leucocytopenia (r = -0.49, p < 0.001). In summary, preexisting cytopenias and hyperferritinaemia indicated long duration of grade ≥3 post-CAR T-cell cytopenias. Prolonged cytopenia may be related to immune remodelling with a shift in the CAR-negative T-cell subsets following CAR T-cell therapy.
•RUNX1 isoform disequilibrium toward RUNX1A and its interaction with MYC:MAX are key in the pathogenesis of trisomy 21–associated ML.•Restoration of RUNX1A:RUNX1C equilibrium and pharmacological ...interference with MYC:MAX dimerization reverses the oncogenic phenotype.
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Gain of chromosome 21 (Hsa21) is among the most frequent aneuploidies in leukemia. However, it remains unclear how partial or complete amplifications of Hsa21 promote leukemogenesis and why children with Down syndrome (DS) (ie, trisomy 21) are particularly at risk of leukemia development. Here, we propose that RUNX1 isoform disequilibrium with RUNX1A bias is key to DS-associated myeloid leukemia (ML-DS). Starting with Hsa21-focused CRISPR–CRISPR-associated protein 9 screens, we uncovered a strong and specific RUNX1 dependency in ML-DS cells. Expression of the RUNX1A isoform is elevated in patients with ML-DS, and mechanistic studies using murine ML-DS models and patient-derived xenografts revealed that excess RUNX1A synergizes with the pathognomonic Gata1s mutation during leukemogenesis by displacing RUNX1C from its endogenous binding sites and inducing oncogenic programs in complex with the MYC cofactor MAX. These effects were reversed by restoring the RUNX1A:RUNX1C equilibrium in patient-derived xenografts in vitro and in vivo. Moreover, pharmacological interference with MYC:MAX dimerization using MYCi361 exerted strong antileukemic effects. Thus, our study highlights the importance of alternative splicing in leukemogenesis, even on a background of aneuploidy, and paves the way for the development of specific and targeted therapies for ML-DS, as well as for other leukemias with Hsa21 aneuploidy or RUNX1 isoform disequilibrium.
Trisomy 21 (+21) is a frequent alteration in acute myeloid leukemias (AML) and constitutive in Down syndrome myeloid leukemia (ML-DS). In this Plenary Paper, Gialesaki et al show that ML-DS blasts are dependent on RUNX1 and reveal that disequilibrium among RUNX1 transcripts with disproportionately increased level of RUNX1A is pivotal for DS leukemogenesis and prominent in sporadic AML. This validates a new model for the early stages of ML-DS development and suggests new therapeutic targets for +21 AMLs in general.
Introduction: Anti BCMA CAR T-cell therapy (CART) is establishing itself as a new standard of care for relapsed and refractory multiple myeloma (RRMM). However, the optimal follow-up strategy post ...CART has yet to be determined. Prior studies of our own and others have emphasized the role of functional imaging in monitoring RRMM patients post traditional chemotherapy, revealing residual disease in up to 50 % of otherwise minimal residual disease (MRD) negative patients (Rasche et al. Leukemia 2019). Whether a similar patchy disease pattern is relevant post CART remains unknown. To address this, we implemented a standardized follow-up program post CART, which includes 18F-FDG PET-CT, diffusion-weighted MRI, and bone marrow aspiration with MRD measurements at our center. Methods: We conducted monthly ambulatory visits and serological testing to monitor patients post CART infusion. Additionally, an extended follow-up visit at three months post CART infusion was performed, encompassing whole body PET-CT, whole body diffusion-weighted MRI, and bone marrow aspiration with MRD testing. Response assessments were based on IMWG criteria. MRI classified focal lesions as vital if they exhibited 1) significant diffusion-restriction, 2) a fat fraction less than 20 %, and 3) homogenous hypointensity on T2-weighted images. MRD measurements were performed with flow cytometry with a sensitivity of at least 10 -5 following the Euroflow recommendations. Results: From December 2021 to July 2023 we treated 37 heavily pretreated patients with idecabtagene vicleucel at our center. These patients had a median of 5 prior lines of therapy. High-risk cytogenetics, extra-medullary disease (EMD), and non-secretory disease were seen in 17, 14 and 6 patients, respectively. At data cut off, the ORR, VGPR and CR rates were 78 %, 27 % and 27 %, respectively. We observed primary refractory disease in 14 % of the patients. Among the 22 patients who underwent extended follow-up examinations at three months, the majority achieved MRD negativity, with only 7/22 (32 %) remaining MRD positive after CART. However, functional imaging revealed a higher number of patients with residual disease, with positive results on PET-CT and DW-MRI in 9/22 (41 %) and 10/22 (45 %) of patients, respectively. Notably, 6 of 22 (27 %) patients displayed active lesions on imaging despite MRD negativity at the iliac crest at this time point. Next, we focused on patients who experienced relapse from CART and sought to determine the necessary technology to detect relapse in this setting. Thirteen patients experienced relapse during the study period, and in 8 (61%) of them, functional imaging was the sole method to detect progressive disease. Only 1 patient transitioned from MRD negative to positive during the follow-up period, while the remaining 4 patients showed a significant M protein increase as defined by the IMWG criteria. Conclusion: In our heavily pretreated RRMM population, functional imaging emerged as a crucial diagnostic tool, detecting relapse alone in more than half of the patients. Our data supports inclusion of cross-sectional imaging in a comprehensive follow-plan after CART.
Background: BCMAxCD3 bispecific antibodies (BsAb) have changed the treatment landscape in relapsed refractory multiple myeloma (RRMM). However, despite high response rates, the majority of patients ...continue to relapse. Infectious complications, mainly viral, represent another challenge with BsAb, seen in up to 90% of patients with half of them requiring hospitalization. Both issues, loss of response as well as infectious complications, can be linked to impaired T-cell function potentially emerging during BsAb therapy. Most BsAbs have been developed as weekly treatments and one hypothesis is that dose dense treatment could overstimulate T cells finally leading to exhaustion and dysfunctionality. Thus, we compared T cell profiles and their functionality from patients with weekly dosing to patients who switched to a monthly schedule due to toxicity or patient request. Methods: We longitudinally biobanked peripheral blood samples of 19 patients of whom 7 switched from a weekly (q1w) to a monthly (q4w) BsAb treatment schedule. Peripheral blood mononuclear cells (PBMCs) were analysed using 16-color flow cytometry, including T-cell subset phenotyping and the exhaustion marker PD1, TIM3, SLAMF6 and KLRG1. Furthermore, CITEseq, scTCRseq, and Seahorse metabolic studies were performed on selected patients. Results: The enrolled patients were heavily pretreated with a median number of 6 prior therapy lines. All patients responded to BCMA BsAb with VGPR or better and were on treatment for >3 month. Only one patient from the q4w cohort relapsed during the study period. High-risk cytogenetics, lines of previous therapy, and penta-refractory disease status were equally distributed between patients on weekly and monthly schedule. Infectious complications occurred in 10/19 (q1w) (52.6%) and 1/7 patients (q4w) (14.2%). First, we addressed the question if a monthly treatment schedule confers less T-cell exhaustion by comparing PD-1 checkpoint expression and T-cell subsets between q1w and q4w administration. Indeed, flow cytometry showed a significant decrease in the CD4+PD1+ (36.9 vs. 21.1% p<0.05, unpaired t-test) and in the CD8+PD1+ (29.44 vs. 16.54% p<0.05) T cells. We also observed a decrease in the frequency of the effector memory subsets (CD45RA-, CD62L-, TEM) of CD4+ (66.19 vs. 49.63% p<0.05) and CD8+ (53.7 vs. 40.32% p=0.09), further supporting a concept in which a treatment free interval leads to recovery of the T-cell system. We also had access to paired samples of five patients who switched from q1w (1 st sample) to q4w dosing (2 nd sample). Here, flow cytometry showed a decrease of exhausted CD4+PD1+ T cells (33.68 vs. 21.76% p=0.046, paired t-test) and CD8+PD1+ T cells (29.2% vs. 15.1% p=0.117), confirming our cross-sectional analysis. Likewise, expression of SLAMF6, a regulator of T cell exhaustion, also decreased from weekly to monthly schedule (SLAMF6/CD4+:20.7 vs. 15.2% p=0.06; and CD8+:17.8 vs. 11.3% p=0.1), but differences were not statistically significant probably due to sample size. Our analysis on T-cell metabolism did not reveal significant differences between weekly and monthly dosing schedules. After strict quality control, three of the paired samples were further analysed using CITE-seq and scTCRseq. In line with our flow results, patients showed a significant decrease in CD8+ pre-exhausted T-cell subset (CD8+TCF-1+CD27+) (3.55 vs. 1.54% p=0.02) and CD8+ terminal differentiated effector memory T-cells (CD8+ TEMRA) (4.66 vs. 3.57% p=0.04) from weekly to monthly schedules, again suggesting recovery of the T-cells after a longer treatment-free interval. We also observed a trend towards decrease in the CD4+ effector memory subset (CD4+TEM, 6.96 vs. 4.89% p=0.07). Interestingly, the number of hyper-expanded T cell clonotypes increased from weekly to monthly dosing in paired samples. This was somewhat unexpected but expansion could be interpreted as reactivation of a few previously exhausted T cells. Conclusions: The optimal dosing schedule of bispecific antibodies has yet to be determined. Our results support a more extended dose schedule to allow T-cell-recovery between BsAb administrations. Whether such a strategy will result in higher antitumor efficacy or reduced susceptibility to infections, has to be evaluated in future studies.
