Waldschmidt, Johannes M

Abstract Recent therapeutic advances, including bispecific antibodies (bsAbs), have improved outcomes for patients with relapsed/refractory multiple myeloma (RRMM). However, early mortality (EM) remains a significant challenge, even in the era of novel immunotherapies. We conducted a multicenter, retrospective analysis of real-world patients with RRMM treated with commercial bsAbs to evaluate the incidence and causes of EM. Between May 2022 and June 2025, a total of 441 patients were treated across 7 countries. EM, defined as death within 12 months of therapy initiation, occurred in 148 patients (34%), with a median time from bsAb initiation to death of 2.87 months (interquartile range, 1.38-6.85). The leading cause of EM was progressive disease (72%), followed by infection (13%); regardless of the cause of death, 82% had active disease at the time of death. Our findings highlight that despite encouraging efficacy, EM remains high among real-world bsAb recipients, emphasizing the need for improved disease control strategies and supportive care interventions.

Abstract In this study, we investigated tumor intrinsic mechanisms of resistance to cevostamab, a bispecific T-cell engager targeting Fc receptor-like protein 5 (FCRL5) and CD3 that has shown promising clinical activity and durable responses in advanced MM. Longitudinal bone marrow aspirates from nine MM patients treated with cevostamab were analyzed. All patients had previously been treated with both immunomodulatory drugs (IMiDs) and proteasome inhibitors (PI) and eight patients had received daratumumab prior to cevostamab, which was, on average, the 6th line of treatment (mean treatment duration 220±273 days). Five of nine patients responded with ≥PR. Whole genome and transcriptome sequencing was performed on sorted CD138+ MM cells at baseline, and whole exome and transcriptome sequencing was performed on five and three samples, respectively, following cevostamab discontinuation. Baseline FCRL5 expression levels did not correlate with clinical response. However, in one non-responder patient, FCRL5 expression increased four-fold after 28 days of treatment. Given the location of the FCRL5 gene on chromosome 1q—commonly amplified in advanced MM—we evaluated the presence of Gain(1q) in our study cohort. All patients whose best response was ≤PR (n=6) harbored a Gain(1q), whereas it was absent in those who achieved VGPR and sCR (n=3). Crucially, one of the patients that achieved sCR was initially treated with 60 mg of cevostamab for one year and then escalated to a higher dose cohort (90 mg) in the second year. At progression, a clonal FCRL5E115A missense mutation (allelic frequency = 56%) and a subclonal FCRL5G455Efs*19(allelic frequency = 5%) frameshift deletion mutation were detected, both absent at baseline. The missense mutation was located on the extracellular domain of FCRL5 targeted by cevostamab. To validate the functional impact of FCRL5E115Aand FCRL5G455Efs*19 on the sensitivity to cevostamab in vitro, we generated a FCRL5 knockout (KO) model utilizing CRISPR-Cas9 technology in OPM2 MM cells. In vitro cevostamab cytotoxicity assay with PBMCs and pan T cells from healthy donors at various effector to target (E:T) ratios confirmed a complete loss of cevostamab sensitivity in our FCRL5KO models. cDNA encoding patient-derived FCRL5 alterations (FCRL5E115Aand FCRL5G455Efs*19) as well as FCRL5wtwere cloned into mammalian expression vector and reintroduced into FCRL5KO cells. Cevostamab sensitivity was reinstated by FCRL5wt expression in KO cells. In contrast, FCRL5E115A conferred complete resistance to cevostamab (p<0,001) and FCRL5G455Efs*19 depicted a significant decline in cevostamab sensitivity relative to FCRL5wt expressing models. Additional experiment utilizing direct stochastic optical reconstruction microscopy (dSTORM) are ongoing to monitor the membrane localization of FCRL5 mutants. By integrating genomic, transcriptomic and functional data with clinical outcomes, this work is the first to report on acquired FCRL5 mutations causing resistance to cevostamab. We hypothesize that the prolonged treatment duration may have contributed to the acquisition of the target mutations and note a subsequent durable sCR response to BCMA-targeted immunotherapy in the same patient, suggesting immune-related factors such as T cell exhaustion, were not drivers of cevostamab resistance. Due to the limited sample size, these findings require validation in larger cohorts. Nonetheless, our findings begin to elucidate tumor intrinsic mechanisms associated with cevostamab resistance.

Abstract Accurate prediction of risk of progression from smoldering (SMM) to active multiple myeloma (MM) is paramount to individualized early therapeutic strategies with minimum risk of overtreatment. Current risk stratification models do not account for evolving biomarker trajectories. We assembled the largest cohort to date of 2,270 SMM patients from six international centers with longitudinal clinical and biological data to train and validate the PANGEA 2.0 risk models. Four evolving biomarkers were significantly associated with shorter time-to-progression: M-protein increase ≥0.2g/dL, involved:uninvolved serum free light chain ratio increase ≥20, creatinine increase >25%, and hemoglobin decrease ≥1.5g/dL. PANGEA 2.0 outperforms established models including the 20/2/20 and IMWG models by more accurately predicting progression (C-statistics=0.69–0.84), even without biomarker history (C-statistics=0.69–0.83) or recent bone marrow biopsy. PANGEA 2.0 is an easy-to-use, open-access tool (https://ghobrial.shinyapps.io/pangea_2_calculator) to improve and individualize SMM risk stratification. Validation tools are available to compare PANGEA 2.0 to established models (https://ghobrial.shinyapps.io/pangea_validation).

Background Chimeric antigen receptor (CAR) modified T-cells are leading to a paradigm shift in the treatment of relapsed/refractory (RR) multiple myeloma (MM). A high proportion of patients treated with CAR T-cells, however, experience prolonged cytopenia, with the mechanism remaining poorly understood. Here, we aimed to explore potential biomarkers that might correlate with cytopenia following CAR T-cell therapy in RRMM. Methods We prospectively collected peripheral blood (PB) of RRMM patients treated with idecabtagen-vicleucel (ide-cel) at the following time points: prior to lymphodepleting chemotherapy (LDC) (baseline), after ide-cel on day 4, 7, 14 and 28, and monthly thereafter. Cytopenia was determined according to the CTCAE version 5.0. Flow cytometry was performed with the following markers: CD45, CD3, CD4, CD8, CD62L, CD45RA, CD19, CD14, CD138, CD38 and a BCMA-CAR-detection marker. Results We included 222 PB samples at different sampling time points from 35 RRMM patients, who were pretreated with a median of 5 therapy lines (range 2-10). All patients were triple-class exposed and, 34 (97%) and 9 (26%) patients underwent autologous and allogeneic stem cell transplant (SCT), respectively. First, we analyzed the relationship between baseline parameters and the duration of grade ≥3 cytopenia after ide-cel (n=35). Notably, patients who received allogeneic SCT or >4 lines of therapy did not develop longer lasting cytopenia than the remaining patients. We found a correlation between the baseline hemoglobin level and the duration of grade ≥3 anemia (r=-0.55, P<0.001). Similarly, low baseline platelet count indicated long duration of grade ≥3 thrombocytopenia (r=-0.48, P=0.003). Moreover, high baseline ferritin level correlated with long duration of grade ≥3 anemia (r=0.52, P<0.001) and thrombocytopenia (r=0.51, P=0.002). Furthermore, the maximum ferritin level after ide-cel was an indicator for long lasting grade ≥3 anemia (r=0.73, P<0.001) and thrombocytopenia (r=0.71, P<0.001). Interestingly, we found significant correlations between the duration of grade ≥3 lymphopenia and baseline CD4+ T-cell frequency (r=0.41, P=0.02), CD8+ T-cell frequency (r=-0.38, P=0.04) and CD4+/CD8+ ratio (r=0.4, P=0.03). Noteworthy, patients with β2-microglobulin level >3.5 mg/l displayed longer duration of grade ≥3 anemia (median: 9 vs 0 day, P=0.007) and neutropenia (median: 43 vs 12 days, P=0.02) compared with the remaining patients, suggesting that high tumor load might be a risk factor for prolonged cytopenia after ide-cel. Second, we divided the follow-up PB samples (n=187) into two groups: early (<d60) and prolonged (≥d60) cytopenia to account for direct toxic effects related to LDC. A high ferritin level was associated with low hemoglobin level (<d60: r=-0.45, P<0.001, ≥d60: r=-0.62, P<0.001) and low platelet count (<d60: r=-0.58, P<0.001, ≥d60: r=-0.81, P<0.001) in both groups. In prolonged cytopenia (≥d60), lymphocyte count correlated with the T-cell count (r=0.7, P<0.001), suggesting that prolonged lymphopenia was mainly attributed to the reduced T-cell count after ide-cel. Of note, the frequency of naïve CD4+ T-cells (CD3+CD4+CD62L+CD45RA+) positively correlated with neutrophil count (r=0.58, P<0.001), hemoglobin level (r=0.37, P=0.03) and white blood cell count (r=0.5, P=0.002) in the ≥d60 group, suggesting that delayed immune reconstruction with reduced naïve CD4+ T-cells might contribute to prolonged cytopenia after ide-cel. Moreover, in the ≥d60 group, lymphocyte count was related with the frequency of CAR+CD4+ (r=-0.58, P<0.001), CAR+CD8+ (r=0.58, P<0.001), CAR-CD4+ (r=-0.72, P<0.001), CAR-CD8+ (r=0.75, P<0.001) T-cells, as well as the ratios of CAR+CD8+/CAR+CD4+ (r=-0.6, P<0.001) and CAR-CD8+/CAR-CD4+ (r=-0.75, P<0.001). Conclusion Here, we present one of the first prospective studies investigating the factors associated with prolonged cytopenia after CAR T-cell therapy in RRMM. High tumor load, preexisting cytopenia and high ferritin level are related with prolonged cytopenia after ide-cel. Delayed immune reconstruction, characterized by reduced naïve CD4+ T-cell count, indicates prolonged cytopenia following ide-cel therapy. Patients with high CD4+/CD8+ ratio are at risk of prolonged lymphopenia (with reduced T-cell count), which may be a risk factor for infectious complications and an issue for subsequent T-cell based immunotherapies.

Abstract Introduction: Novel substances such as the next generation IMiD pomalidomide or the recently approved next generation proteasome inhibitor carfilzomib (Cfz) have considerably expanded our treatment options in MM, both of them influencing multiple myeloma (MM) interaction with bone marrow stroma cells (BMSCs), that provides an interesting target for anti MM therapy. More compounds directed at this disease critical crosstalk are currently under investigation, however the development of novel drugs remains inefficient, displayed by a substantial drop out rate of the 376 preclinical single agents tested since 1961 (Rongvaux, Annu Rev Immunol. 2013; Schüler, Expert Opin Biol Ther. 2013; Kortüm, CLML 2014). Our focus in the projected presented here was to develop a novel bone-derived in vitro 3D co-culture model specifically adapted to mimic the BM niche to more closely study the role of bone and BM bystander cells and to perform more reliable ex vivo compound screening in MM. Methods: Previous 2D models were compared to a novel 3D co-culture model (agarose matrix interlayer, 100 microwells/cm², 1.5mm in depth, permeable for oxygen+cytokines, but not for BMSCs utilizing U266, RPMI-8226, OPM-2 and primary BM patient (pt) cells, with and without HS-5 vs. M210B4 stroma support (Fig. A + Fig. B.a. for pt characteristics). Analyses covered Trypan Blue, Annexin/PI, MTT, FACS, cell cycle analyses and H2B-mCherry/cytochrome c-GFP assays (Udi, Br J Hematol. 2013). In a next step, primary bone-derived stroma cells were acquired from bones of C57BL/6 J mice. Bones were flushed, digested and FACS sorted in order to acquire single BM and bone bystander cell subtypes (MSPCs [mesenchymal stem and progenitor cells], endothelial cells, osteoblasts, PAS [PDFGRalphaSca1] and CaRs [CXCL12-abundant reticular cells]) which were then compared to HS-5 and M210B4 with regard to growth support, cytokine secretion and protection from anti-MM substances. Results: MMCLs and pt specimens were cultured at different concentrations (10 vs. 100 cells per microwell) with and without M210B4 demonstrating a growth advantage with vs. without M210B4 (Fig. B.b). Liquid overlay technique allowed cluster formation of pt specimens leading to more reliable propagation of pt material for up to 20d of culture. Apoptotic changes were assessed by confocal microscopy of RPMI8226 co-expressing fluorescently labelled histone 2B-mCherry (red) and cytochrome c-GFP (green) as indicators of late and early apoptosis. Comparing BMSCLs with regard to their MM growth support capacities, human HS-5 proved even more beneficent than M210B4 stroma (Fig. B.b). Phenotypic analyses of pt specimens showed decreased CXCR4 expression with vs. without BMSCs suggesting a dynamic regulation of homing molecules. The model was then used as an ex-vivo platform allowing both cytotoxicity and cell cycle analyses for the combination of bortezomib (Btz) vs. Cfz with ARRY-520 (kinesin spindle protein inhibitor). Btz (10nM) and Cfz (20nM) proved significantly cytotoxic compared to the control (U266 and pt specimens, respectively) after 48h of single agent treatment Fig.B.c). Compared to Btz (10nM, B10) and Cfz (20nM, C20) as single agents, the additional combination with ARRY-520 showed stronger additive cytotoxicity for Btz (A5+B10, median: 37.5% vs. 13.1%) than for Cfz (A5+C20, 38.6% vs. 34.1%). To note, the model could also be utilized for more profound analyses as depicted for G2/M cell cycle studies in Fig.B.d. 5nM ARRY-520 (A5) led to significant accumulation of OPM-2 cells in G2/M arrest after 48h of treatment confirming prior analyses (Hernández-Garcia Blood Suppl 4710,2014; Fig. B.d). Co-culture studies with different subsets of BM and bone-derived bystander cells are currently ongoing and will be presented at the meeting (Fig. B.e). Conclusions: More complex, 3D bone-derived high-throughput in vitro models are urgently needed to better predict the potency of preclinically tested agents and to better estimate the likelihood of their later clinical adoption into phase I-II trials. With this work, we provide an innovative model which reflects the BM microenvironment as a crucial predictor for in-vivo sensitivity as shown for ARRY-5200. This ex-vivo approach helps to better incorporate MM growth support by bone and BM-derived bystander cells and thus depicts a valid tool to better characterize the role of the BM niche in myeloma. Figure 1. Figure 1. Disclosures Engelhardt: Deutsch Krebshilfe: Other: grant. Wäsch:German Cancer Aid: Research Funding; Comprehensiv Cancer Center Freiburg: Research Funding; Janssen-Cilag: Research Funding; MSD: Research Funding.

Abstract Introduction: In the past decade, substantial progress has been made in the understanding of multiple myeloma (MM) cell biology and its interaction with the bone marrow microenvironment (BMM). Binding of MM cells to BM stroma cells (BMSCs) alters the expression of SDF-1α and its receptor CXCR4, leading to the secretion of anti-apoptotic cytokines, promoting tumor growth, drug resistance and migration. MM cancer stem cells migrate to endosteal BM niches, where they escape therapies in a quiescent state causing relapse in the course of the disease. The development of novel agents that aim to target the MM and BMM interaction includes drugs as promising as 2nd and 3rdgeneration IMIDs or proteasome inhibitors. Despite these profound advances, the failure rate of preclinically proven cytotoxic single substances is sizeable, as preclinical models often lack the biological, genetic, etiological and immunological properties of the disease (Schüler, Expert. Opin. Biol. Ther. 2013; Kortüm. CLML 2014; Rongvaux. Annu Rev Immunol 2013). Methods & Results: We have previously demonstrated that BM interaction and homing to niches, mediated by the adhesion molecules CXCR4, CD49d and CD44, protect MM cell lines (MMCL) and primary plasma cells (PC) from the cytotoxic effect of anti-MM agents, such as bortezomib (Bor), vorinostat (Vor) and pomalidomide (Pom). Our in vitro and in vivo observed cytotoxic effects from Bor, Vor and Pom confirmed their potent cytotoxicity, whereas cocultivation with M2-10B4 substantially reduced apoptosis and induced tumor protective effects. Additional treatment with the CXCR4 inhibitor AMD3100 blocked CXCR4 in coculture, but left CD49d, CD44 and CD11a widely unchanged. Toxic or therapeutic effects from AMD3100 monotherapy were excluded for the doses used. Comparison of the CXCR4 antibody (ab)-clones 12G5, 44717 and 4G10 revealed that AMD3100 treatment of U266 cells reduced CXCR4 expression with use of 12G5 and 44717, whereas binding of both FITC- and PE-coupled 4G10 was not influenced, making the latter the most reliable for CXCR4 analysis. Use of image cytometry (IC) allowed accurate visualization of co-localisation of CXCR4 expression both on the cell surface and within the cytoplasm of MM cells. IC correlated with flow cytometry-determined CXCR4 expression and allowed the detailed assessment of treatment studies with and without anti-MM agents and AMD3100. Of note, AMD3100 resensitized MM cells to Bor, Vor and Pom (Waldschmidt. Blood 2012:2450), whereas carfilzomib (Cfz) reduced CXCR4 expression in MMCL and could not be antagonized by stroma coculture. Cfz sensitivity was not increased by adding AMD3100 (Simon. Blood 2013:3851). These preclinical studies need additional adaptation to the clinical setting in order to surpass prior drug failure rates, and there is a need to develop more broadly available and better predictive preclinical systems. Therefore, we are currently assessing a 3D co-culture MM model composed of agarose matrix interlayers, based on a novel liquid overlay technique. This model has been specifically adapted to MM cell and BM component interactions as described (Udi. BJH 2013; Zlei. Exp Hematol 2007; Schüler. EOBT 2013). MM cells are cultivated in conical microwells of a non-adherent agarose matrix after BMSCs were plated on the bottom of each plate, allowing the diffusion of soluble cytokines but no direct contact between BMSC and MMCL. Therein, we are presently testing novel anti-MM substances in comparison to our standard-coculture system. Conclusion: Targeting microenvironmental mediators, like SDF-1α and CXCR4, is a promising approach to expand the choice of antimyeloma agents and amplify the effects of established antimyeloma drugs, as previously shown by us and others for the combination of AMD3100 and Bor or Pom. However, as our knowledge on MM and its BMM has dramatically increased a great effort has been made in the preclinical testing of promising new anti-MM agents, and more complex high-throughput in vitro models are urgently needed to better predict the potency of these substances in order to reduce dropouts in clinical trials. We hereby provide a novel approach which better reflects the spatial growth of human MM samples in BMSC coculture, and more closely mimics the growth and proliferation of human MM clones in vivo. Disclosures No relevant conflicts of interest to declare.