Sensitive, scalable and affordable measurable residual disease (MRD) assays are needed to guide treatment decisions in acute myeloid leukemia (AML), particularly around allogeneic hematopoietic stem cell transplantation (allo-SCT). Next-generation sequencing (NGS)-based MRD assays offer broad applicability and high sensitivity, but remain too costly for routine use in resource-limited environments. We developed a cost-efficient, sensitive NGS MRD assay utilizing single-molecule molecular inversion probes (smMIPs) targeting 92 genomic loci in 33 AML driver genes and applied it to 93 AML patients in remission prior to allo-SCT. MRD positivity, defined as the presence of ≥ 1 non-DTA (DNMT3A, TET2, ASXL1) gene variant with ≥ 0.5% variant allele frequency (VAF), was associated with significantly shorter post-transplantation overall survival (OS; p < 0.001). In multivariable analysis, NGS-based MRD detection remained an independent predictor of inferior OS (hazard ratio 4.58; p = 0.002). Conditioning intensity did not associate with outcome of MRD-positive patients in this retrospective cohort. Mutations at diagnosis and pretransplantation showed variable concordance across genes, and consistently lower VAFs at the later timepoint. In three patients, multiple low-VAF clustered variants in RUNX1 and TET2 were detected pre-transplantation, potentially indicating treatment-induced mutagenesis. These findings demonstrate that a broadly applicable, smMIP-based NGS MRD assay can provide clinically relevant risk stratification before allo-SCT in AML, while its low library preparation costs of approximately 8€ per sample may facilitate wide implementation in routine practice and allow more patients to receive MRD-directed therapeutic interventions.

Given the selection of elderly patients with AML in first complete remission (CR1) the advantage of consolidation with allogeneic hematopoietic cell transplantation (HCT) over chemotherapy is still unclear. Newly diagnosed AML patients in CR1 aged 60-75 years were registered and a donor search initiated. After one consolidation cycle, patients with a matched donor were randomized to HCT with fludarabine/low-dose total body irradiation and cyclosporine/mycophenolate mofetil immunosuppression or conventional non-HCT. Primary outcome was restricted mean leukemia-free survival (RM-LFS) up to 5 years. Between 2010 and 2017, 245 patients (median age 67 years) were registered at CR1. After one consolidation, 26.9% of patients failed inclusion criteria. Of the 179 (73%) patients still on study, 75.4% had an HLA identical donor. Ten ineligible patients were excluded, and 125 randomized to HCT (N=83) or non-HCT (N=42). The primary outcome RM-LFS up to 5 years was 24.5 months (95% confidence interval [CI]: 18.9-30.1) in the HCT and 15.6 months (95% CI: 10.4-20.8) in the non-HCT arm (P=0.022) due to a decrease in cumulative relapse incidence from 91.1% (95% CI: 80.7-100.0) after non-HCT to 37.8% (95% CI: 27.2-48.4) after HCT (P<0.0001). The secondary endpoints RM-OS up to 5 years was 27.8 months (95% CI:22.3-33.2) in the HCT as compared to 28.6 months (95% CI: 22.2-35.0) in the non-HCT arm; non-relapse mortality at 5 years was 33.4% (95% CI: 23.0-43.9) with HCT and 0% without. In older patients with AML in CR1 5-year RM-LFS is better with HCT than with non-HCT consolidation treatment. The long-term RM-LFS benefit did not translate into a better RM-OS during the study period.

BACKGROUND:Apheresis procedure of autologous lymphocytes competent for proliferation and expansion is a crucial step in the production of chimeric antigen receptor (CAR) T-cells. Previous therapies or disease status prior to collection may negatively impact the collections. STUDY DESIGN AND METHODS:We performed a retrospective analysis with the aim to determine cellular factors in association with the collection of autologous T-cells and subsequent CAR T manufacturing toward tisagenlecleucel (tisa-cel). Between February 2019 and February 2022, 63 collections of 54 patients were performed for subsequent therapy with tisa-cel. RESULTS:We observed no difference in median CD3+ cell yields according to the number of prior therapy lines (>3 vs. ≤3, p = .335), prior treatment with bendamustine (p = .954) or marrow infiltration (p = .634). Fifty-six collections were sent for manufacturing, of which 22 (39%) resulted in manufacturing failures, namely terminations (n = 12) or out-of-specification events (n = 10). Collections resulting in manufacturing failures yielded significantly lower CD3+ (p = .005), CD3+CD4+ (p = .044), and non-senescent CD3+CD27+CD28+ (p = .003) counts. Multivariable analysis identified the absolute number of CD3+CD27+CD28+ cells as relevant, with a calculated cut-off of ≥34.58 × 108 CD3+CD27+CD28+ cells for 89.5% probability of successful CAR T-cell production. DISCUSSION:In summary, we report a positive influence of a higher number of non-senescent Τ-cells on successful manufacturing. Further analyses are required to determine measures for further optimization of collection outcomes.

Introduction Relapse remains the main reason for poor outcomes of patients (pts) with acute myeloid leukemia (AML). Allogeneic hematopoietic stem cell transplantation (HSCT) is the preferred post-remission treatment in pts at high risk of relapse. The presence of measurable residual disease (MRD) after intensive frontline treatment or before or after HSCT has emerged as an important predictor of relapse. Various methods are used for MRD detection, including flow cytometry and molecular assays detecting individual gene mutations (mut) such as NPM1mut. Next generation sequencing (NGS)-based assays covering multiple genes commonly mutated in AML may allow sensitive MRD detection in a broad range of pts with diverse genetic characteristics. Methods We established a targeted, error corrected NGS MRD assay utilizing single-molecule molecular inversion probes (smMIPs). The smMIP protocol uses a hybridization-capture approach to selectively enrich and amplify both DNA strands of 92 regions in 32 genes in a single reaction. Unique molecular identifiers (UMIs) facilitate computational correction of sequencing errors to enable reliable detection of variants ≥0.5% variant allele frequency (VAF), with the additional advantage of relatively low reagent and sequencing costs of < $100 per sample. The smMIP assay was used to evaluate MRD status on pre-HSCT bone marrow samples from 98 AML pts who received an HSCT in first (n=83) or second (n=15) complete remission with or without count recovery. Median age at HSCT was 59 (range 20-75) years, 42% were female; 70% had de novo AML, 19% had an antecedent myeloid neoplasm and 11% had AML post cytotoxic therapy. European LeukemiaNet (ELN)-2022 risk groups at diagnosis were favorable in 11%, intermediate in 33%, adverse in 20% and unknown in 22%. All pts had received intensive induction chemotherapy. Twenty-nine percent received a myeloablative conditioning regimen (MAC), while 23% received reduced intensity conditioning (RIC) and 48% a non-myeloablative (NMA) conditioning regimen. Donors were matched related (18%), haploidentical (6%), or matched (55%) or mismatched (21%) unrelated. The median follow-up for pts alive was 34.8 months. Results Overall, we detected 190 variants in 71 (72%) of 98 pre-HSCT remission samples. The most commonly affected genes were DNMT3A (in 37% of pts), TET2 (21%), PPM1D (12%), IDH2 (10%) and TP53 (5%). Twenty-nine percent of pts had only variants affecting the clonal hematopoiesis-related genes DNMT3A, TET2 or ASXL1 (‘DTA‘), while 44% of patients had ≥1 variant involving a non-DTA gene. MRD-positive pts, defined as those with ≥1 non-DTA variant, had similar baseline and HSCT-related characteristics (includingage, sex, ELN risk group, de novo vs. secondary AML) compared to pts without non-DTA variants. Overall survival (OS) was significantly inferior in NGS MRD-positive pts compared to those with no non-DTA variant (Figure A, 5-year OS, 49% vs. 85%, P=.011). MRD-positive pts also had a non-significant higher cumulative incidence of relapse at 5 years (46% vs. 24%, P=.14), while non-relapse mortality was similar in both groups (P=.72). We next performed multivariable analyses, considering variables associated with OS at an univariable P<.10 (MRD status, patient sex, therapy-related AML, conditioning intensity), and using stepwise backward variable selection to identify factors significantly associated with OS (Figure B). In the final multivariable model, pre-HSCT MRD detection remained associated with inferior OS (hazard ratio 2.51; 95% confidence interval, 1.07 - 5.91; P=.035). Female sex associated with favorable OS, while myeloablative conditioning (MAC) showed a borderline significant association with superior OS. Of note, we did not detect an interaction between conditioning intensity and detection of MRD with regard to OS, i.e. the prognostic association of MRD detection was not different between pts receiving myeloablative or less-intensive conditioning. However, this analysis was limited by the small patient number. Conclusion Detection of MRD by targeted NGS using the smMIPs approach provides prognostic information in pts undergoing allogeneic HSCT, including those without established molecular markers such as NPM1mut. This method is applicable to a large proportion of AML pts and is cost-efficient for broad clinical use. S.M.K. and T.H., and M.J. and K.H.M. contributed equally to this abstract.

F I G U R E 2 Outcomes according to high (≥1%) or low (<1%) NPM1/ABL1 measurable residual disease (MRD) levels.(A) Time to relapse in relapsing patients (median time to relapse in patients with 0.01%-<1% NPM1/ABL1 burden at hematopoietic stem cell transplantation (HSCT): 159 days; median time to relapse in patients with ≥1% NPM1/ABL1 burden at HSCT: 75 days, (B) cumulative incidence of relapse and (C) overall survival.

B cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T cells revolutionized the treatment of relapsed/refractory multiple myeloma (RRMM). However, data on cellular (CAR) T cell dynamics and the association with response, resistance or the occurrence of cytokine release syndrome (CRS) are limited. Therefore, we performed a comprehensive flow cytometry analysis of 27 RRMM patients treated with Idecabtagene vicleucel (Ide-cel) to assess the expansion capacity, persistence and effects on bystander cells of BCMA-targeting CAR T cells. Additionally, we addressed side effects, like cytokine release syndrome (CRS) and cytopenia. Our results show that in vivo expansion of CD8 + CAR T cells is correlated to response, however persistence is not essential for durable remission in RRMM patients. In addition, our data provide evidence, that an increased fraction of CD8 + T cells at day of leukapheresis in combination with successful lymphodepletion positively influence the outcome. We show that patients at risk for higher-grade CRS can be identified already prior to lymphodepletion. Our extensive characterization contributes to a better understanding of the dynamics and effects of BCMA-targeting CAR T cells, in order to predict the response of individual patients as well as side effects, which can be counteracted at an early stage or even prevented.

Introduction: Even after allogeneic hematopoietic stem cell transplantation (HSCT) relapse remains a main driver of mortality in acute myeloid leukemia (AML) patients (pts). In overt relapse, treatment options are limited and survival is significantly shortened. Early detection of impeding relapse by monitoring measurable residual disease (MRD) could improve outcomes by identifying pts that would benefit from preemptive treatment. Most studies analyzing molecular MRD used mutation (mut) panels and did not differentiate between different genes. However, distinct genes may provide distinct prognostic relevance, as shown for ASXL1, DNMT3A, or TET2 (“DTA”) mut, which do not function as MRD markers after chemotherapy. Mut in IDH1 and IDH2 occur in up to 20% of AML pts, and cluster at three hotspots, making them potential easy targets for sensitive PCR-based MRD detection. So far, no larger study analyzed their relevance for MRD detection after HSCT. Methods: We screened 462 AML pts for IDH mut by NGS (MiSeq platform, Illumina) or Sanger sequencing and detected IDH mut in 100 pts (IDH1 R132: n=44; IDH2 R140: n=37; IDH2 R172: n=18; IDH1 R132 & IDH2 R140: n=1). At HSCT, IDH mut pts were in first complete remission with or without count recovery (CRc, 70%), second CRc (15%), or relapsed/refractory (15%). Median age at HSCT was 64 (range 20-76) years (y). Conditioning regimens were myeloablative (10%), reduced-intensity (30%) or non-myeloablative (60%). MRD analyses were performed by custom designed mut specific digital droplet PCR probe assays as published previously. IDH MRD positivity (MRDpos) was defined as VAF ≥ 0.05%. Median follow up after HSCT was 3.6 y. Results: Compared to all others,presence of IDH1 or IDH2 mut at diagnosis did not significantly associate with outcomes (cumulative incidence of relapse [CIR] P=.13, overall survival [OS] P=.40). In CRc prior to HSCT (n=66), 59% of IDH1 mut, 81% of IDH2 R140 mut and 38% of IDH2 R172 mut pts remained IDH MRDpos, which did not associate with CIR (P=.17) or OS (P=.80). In CRc after HSCT, the IDH MRD status was available for 57 pts (IDH1 R132: n=25; IDH2 R140: n=26; IDH2 R172: n=7) with a median of 4 samples/pt (range 1-21). 34% of IDH mut pts relapsed during follow-up, and all pts with available relapse material (n=16) were positive for their known IDH mut (median VAF at relapse 18.7%, range 0.09-29%). In all relapsing pts with samples available, relapse was preceded by at least one MRDpos sample (median VAF in first pos sample 0.23, range 0.07-19.9%). Median time from first MRDpos sample to overt relapse was 51 (range 8-341) days (IDH1: median 40.5 [range 8-119] days, IDH2: median 91 [range 13-341] days). Of the pts in ongoing CRc with post-HSCT samples available (n=40), 3 pts had 1 MRDpos sample (IDH1: 2 pts; IDH2 R140: 1 pt) and 1 pt had two MRDpos samples (IDH2 R172). In 2 pts these positive samples occurred directly after HSCT and converted to MRD negativity (MRDneg), 1 pt died from an acute infection 96 day after testing MRDpos and 1 pt converted to MRDneg after suffering from grade 3 acute graft-versus-host disease (GvHD). ROC curves showed that the IDH1 and IDH2 MRD status after HSCT were highly predictive of relapse within the next 56 days (AUCIDH1=.98 and AUC IDH2=.93) and 84 days (AUC IDH1=.91 and AUC IDH2=.90). Of note, ROC comparison showed that IDH1 and IDH2 MRD was not inferior in predicting relapse than the NPM1 MRD status within 56 days (AUCNPM1=.95, compared to IDH1: P=.19 and IDH2: P=.81) and 84 days (AUCNPM1=.81, compared to IDH1: P=.27 and IDH2: P=.35) in NPM1 mutated pts. Pts with at least one IDH MRDpos sample within the first year after HSCT had a significantly higher CIR (P<.001) and shorter OS (P=.001). This was also seen when IDH1 and IDH2 mut were regarded separately (IDH1: CIR P<.001 and OS P=.005; IDH2: CIR P<.001 and OS P=.07). Conclusion: Neither the IDH mut status at diagnosis nor IDH-based MRD in CRc prior to HSCT associated with outcomes.In contrast, after allogeneic HSCT, IDH1 and IDH2-based MRD reliably predicted relapse in AML pts and was not inferior to NPM1 mut based MRD. Our data indicates that all IDH mut only function as MRD marker after allogeneic HSCT, which resembles mut associated with clonal hematopoiesis, like DTA. This should be taken into account when assessing MRD in clinical practice. The conversion of MRDpos to MRDneg in pts early after HSCT or after suffering GvHD implies that immunological effects can eradicate MRD after HSCT.

Background: In acute myeloid leukemia (AML), clonal hematopoiesis (CH)-associated mutations usually appear early in leukemogenesis, and often persist in complete remission following chemotherapy. The presence of residual CH at the time of a consolidating allogeneic hematopoietic stem cell transplantation (HSCT) has no negative impact on outcomes of AML patients (pts). Here, we analyzed the prognostic impact of measurable residual CH (MRCH) early after HSCT. Results: In remission before HSCT 83% of pts remained MRCHpos, which did not associate with a higher cumulative incidence of relapse (CIR, P=.19) or shorter relapse-free survival (RFS, P=.08, with a trend for higher CIR in patients without persisting MRCH). Fifty-one (35%) pts relapsed after HSCT. In 29 of these pts material at relapse was available and showed that the CH mutation detected at diagnosis was also present in all but one relapse samples (median VAF at relapse of 11.8 [range 0.2-68.3]%). Of those, 25 pts had at least 1 post-HSCT sample available prior to relapse. In 22/25 pts, impeding relapse was preceded by detectable MRCH (first positive sample with a median VAF of 0.25 [range 0.06-12.3]) at a median of 55 days prior to morphologic relapse. In two non-converting (conversion from MRCHneg to MRCHpos) patients who relapsed, both with SRSF2 mutations, the last samples 132 and 35 days prior to relapse were MRCHneg. One non-converting patient suffered from an extramedullary relapse without bone marrow involvement. A total of 19% of pts died without relapse. Of the pts alive in remission, the majority remained MRCHneg (Figure 1A). At all evaluated timepoints post-HSCT, we observed a significantly higher CIR and shorter RFS for AML pts in morphologic remission who had detectable MRCH: at day 28 after HSCT (P=.01, and P<.001, respectively), day 100 after HSCT (P<.001, and P<.001, respectively, Figure 1B), day 180 after HSCT (P=.003, and P=.002, respectively), and day 360 after HSCT (P=.001, and P=.04, respectively). While the risk of relapse remained high in MRCHpos pts irrespective of the timepoint of assessment during post-HSCT follow-up (46-66% after 3 years), the 3-year risk of relapse in MRCHneg pts continuously decreased with time after HSCT (28% at day +28, 13% at day +100, 5% at day +180, 0% at day +360 in MRCHneg pts, respectively). Conclusions: In contrast to the absent prognostic relevance of persisting MRCH in remission before HSCT, the presence of detectable MRCH after HSCT was linked to a higher CIR and shorter RFS at every evaluated timepoint early after HSCT. The risk of relapse in MRCHneg pts continuously decreased within the first year after HSCT: the longer pts remained MRCHneg the less likely relapse occurred. Our data suggests MRCH as a feasible marker for residual disease assessment in AML pts in morphologic remission after allogeneic HSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction Non-relapse mortality (NRM) remains a relevant risk for acute myeloid leukemia (AML) patients (pts) undergoing allogeneic hematopoietic stem cell transplantation (HSCT). CT-defined body composition parameters are increasingly recognized as modifiable risk factors in cancer pts. Here, quantity and quality of skeletal muscle areas (SMA) as surrogate parameters for sarcopenia, and the amount of adipose tissue are associated with NRM. Large analyses in pts with hematological malignancies are scarce. We addressed this topic here in AML pts receiving HSCT. Methods We retrospectively analyzed 429 AML pts who received routine abdominal CT scans to exclude infectious foci up to 7 weeks prior to myeloablative (25%), reduced intensity (15%), or non-myeloablative (NMA, 60%) HSCT. Median age at HSCT was 59 (range 17-76) years (y) and pts were in 1st complete remission with/without blood count recovery (CRc, 65%), later CRc (17%), or relapsed/refractory (18%). European LeukemiaNet (ELN22) risk at diagnosis was favorable 22%, intermediate 27% and adverse 51%. Body composition parameters were determined in CT on the level of the third lumbar vertebra. SMA and visceral adipose tissue (VAT) were measured on a predetermined range of radiodensity expressed in Hounsfield Units using ImageJ software. SMA was divided by the body height squared to calculate skeletal muscle index (SMI). Muscle quality was defined by intramuscular adipose tissue content (IMAC), measured using the muscle radiodensity, divided by that of the subcutaneous adipose tissue - with a higher IMAC indicating lower muscle quality due to a higher adipose tissue portion. Cut-offs were determined using ROC curves in a test set (1/3 of pts) and validated in a confirmation set (2/3 of pts) randomly partitioned by sex. Results Pt characteristics and outcomes did not differ between the test and confirmation set (cumulative incidence of relapse [CIR] P=.52, NRM P=.79, OS P=.20). Of all pts, 69% were sarcopenic, defined by lower SMI (cut-off male [m] 52.36cm²/m²; female [f] 40.38cm²/m²). Pts with sarcopenia had a lower body mass index (BMI) at HSCT (P<.01), a lower IMAC (P<.01) and a lower VAT (P<.01). 13% of pts were viscerally obese, defined by higher VAT (cut off m 159.85cm2, f 106.26cm2). Pts with visceral obesity were older (P=.03), had a higher BMI at HSCT (P<.01), a higher SMI (P<.01) and higher IMAC (P<.01). They also had a higher incidence of a complex karyotype (CKT, P=.01), more SRSF2 mut (P=.03) and more often developed a chronic graft versus host disease (GvHD, P=.01) after HSCT. 69% of pts had low muscle quality, defined by higher IMAC. They were older (P<.01), had a higher BMI at HSCT (P<.01), a higher VAT (P<.01) and a higher SMI (P<.01). They also less frequently had de novo AML (P=.01), a higher incidence of a CKT (P=.03), more SRSF2 mut (P=.03) and worse ELN22 risk (P=.03). Pts with a high IMAC had a higher HCT-CI score (P=.02), more often received NMA HSCT (P<.01) and more often received HSCT from unrelated (P<.01) or mismatch donors (P<.01). After HSCT, they more often developed acute (P<.01) and chronic GvHD (P=.04). Outcomes did not differ significantly according to the presence of sarcopenia (CIR P=.14, NRM P=.08, OS P=.20). Viscerally obese pts had similar CIR (P=.29), but a higher NRM (P=.03) which translated in a trend for shorter OS (P=.06). Pts with high IMAC had similar CIR (P=.25), but higher NRM (P<.01) and shorter OS (P<.01). In multivariate analysis, low muscle quality (i.e. higher IMAC) and visceral obesity (i.e. higher VAT) remained prognostic for higher NRM (Hazard ratio [HR] 3.40, 95% confidence interval [CI] 1.67-6.94, P<.01 and HR 1.85, 95% CI 1.05-3.28, P=.04) after adjustment for ELN22 risk and a higher IMAC for shorter OS (HR 0.53, 95% CI 0.35-0.8, P<.01) after adjustment for remission status at HSCT. As the IMAC significantly associated with OS, but differed according to age and BMI at HSCT, we performed additional subgroup analyses. Here, a high IMAC had the highest impact on OS in younger pts (≤60y P<.01, less in pts >60y [P=.04]) and pts with a BMI ≤ 25kg/m2 at HSCT (OS P<.01), with a lower impact in overweight (P=.07) and none in obese pts (P=.40). Conclusions In AML pts undergoing HSCT, visceral obesity associated with higher NRM and low muscle quality with higher NRM and shorter OS. Strategies to improve mobilization, physical activity and nutrition during treatment should be investigated to improve outcomes of these patients.

Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment of relapsed/refractory multiple myeloma (RRMM). Robust variables that predict long-term response are currently missing. Limited data are especially available on the impact of bridging therapies on manufacturing and outcome. We conducted a longitudinal single-cell multi-omics study to identify factors that predict response to BCMA-directed CAR T cells. Changes in the immune microenvironment associated with response were analyzed as well as the impact of prior bridging therapy with bispecific antibodies on subsequent CAR T cell manufacturing and outcome. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 29 consecutive MM patients treated with commercially available anti-BCMA CAR T cells on the day of leukapheresis as well as days 30 and 100 after CAR T cell infusion. PBMCs were subjected to single cell RNA, T-cell receptor (TCR) and B-cell receptor (BCR) sequencing. A custom panel of 57 oligonucleotide-coupled antibodies was used to study surface proteomics. Downstream analyses were performed with Seurat. Differences in cellular compositions at all three time points were analyzed with scCODA. To analyze CAR T cell functionality, CAR T cells from peripheral blood were isolated 7 days after infusion and subjected to an in vitro cytotoxicity assay after expansion and stimulation. Patients were grouped based on their best response following CAR T cell infusion (CR: n=12, non CR: n=17). Results: In total, 375,338 cells were sequenced (median 7246 cells/sample, range 1,569-10,972 cells) and 354,878 cells (94.5%) passed quality assessment. Quantitative and qualitative differences in the cellular composition of peripheral blood between CR and non CR patients were detected at the time of leukapheresis as well as on days 30 and 100 following infusion. CR patients harbored significantly more CD8+ effector memory T cells (TEM) at leukapheresis and less NK cells on day 30 after therapy compared to non CR patients. Regulatory T cells isolated at the time of leukapheresis from non CR patients exhibited significantly higher surface protein levels of CXCR3, CD40, CD95 and KLRG1 (p<0.015, respectively) that have been associated with T cell senescence and impaired tumor immunity. No significant differences in cell numbers between CR and non CR were detected on day 100 after CAR T cell infusion. However, single cell TCR analysis revealed an increasing diversity in the TCR repertoire over time in patients with CR, while Shannon diversity decreased from leukapheresis over day 30 to day 100 in non CR patients (p=0.004). The prior administration of the bispecific antibody teclistamab had no significant impact on the quantitative cellular composition at the time of leukapheresis. However, termination of manufacturing in the first attempt occurred in all patients with a close proximity of teclistimab administration and apheresis. Differential gene expression analysis showed that the application of teclistamab was associated with impaired T cell activation and exhaustion indicated by upregulation of e.g. CTLA4, TIGIT, LAG3 and GZMK. After discontinuation of teclistamab (median 4 weeks) and successful manufacturing of CAR T cells, we found no significant differences for in vitro cytotoxicity and in vivo expansion of CAR T cells. CAR T cells isolated at day 7 post-infusion from patients in CR, non CR or with prior teclistamab exposure, effectively eliminated MM cells (U-266). Tracking of single CAR T cells over time showed that the majority of CAR+ cells were CD8+ TEMs regardless of remission achievement or prior teclistamab exposure. Conclusion: We demonstrate that differences between MM patients achieving a CR and patients with suboptimal response upon anti-BCMA CAR T cell therapy can already be identified at the time of leukapheresis. Long-term changes associated with CR include a diversification of the TCR repertoire. Successful CAR T cell manufacturing is hampered by exposure to bispecific antibodies but can be successfully achieved by allowing for a wash-out phase of ca. 4 weeks.

Background:Red blood cell distribution width (RDW) is calculated in every blood count test and reflects variability in erythrocyte size. High levels mirror dysregulated erythrocyte homeostasis and have been associated with clonal hematopoiesis as well as higher mortality in several conditions.We aimed to determine the impact of preprocedural RDW levels on functional outcomes after transcatheter aortic valve implantation (TAVI). Methods:In this single-center retrospective study, we analyzed 176 consecutive patients receiving TAVI between 2017 and 2021. RDW upper limit of normal was < 15 %. Patients were stratified according to preprocedural RDW as having normal or elevated values. We assessed all-cause-mortality and a composite endpoint comprising cardiovascular/ valve-related mortality and cardiovascular, valve-related and heart failure hospitalization at 1 year. Results:43 patients (24.4 %) had RDW ≥ 15 %. There were significant baseline differences between groups (Society of Thoracic Surgeons - Predicted Risk of Mortality score 3.18 %[interquartile range 1.87-5.47] vs. 6.63 %[4.12-10.54] p < 0.001; hemoglobin 13.2 g/dL[11.8-14.1] vs. 10.4 g/dL[9.8-12.2], p < 0.001, RDW-normal vs. RDW-high, respectively). Age was not distinct (80.2 years [77.5-84.1] vs 81.2[71.3-84.7], p = 0.78). 1-year-all-cause mortality was not different (7.9 % vs. 9.4 %, p = 0.79). The RDW-high group showed markedly higher NT-proBNP levels after 1 year (647 ng/ml[283-1265] vs. 1893 ng/ml[744-5109], p = 0.005), and experienced more clinical endpoints (hazard ratio 2.57[1.28-5.16] for the composite endpoint, p = 0.006). RDW remained an independent predictor of the composite endpoint when accounting for all baseline differences in multivariable regression. Conclusion:Elevated preprocedural RDW identifies patients at risk for impaired functional outcome after TAVI and may represent a useful low-cost parameter to guide intensity of outpatient surveillance strategies.