Ex vivo propagation in a novel 3D high-throughput co-culture system for multiple myeloma

PURPOSE: Multiple myeloma (MM) remains an incurable hematologic malignancy which ultimately develops drug resistance and evades treatment. Despite substantial therapeutic advances over the past years, the clinical failure rate of preclinically promising anti-MM drugs remains substantial. More realistic in vitro models are thus required to better predict clinical efficacy of a preclinically active compound. METHODS: Here, we report on the establishment of a conical agarose 3D co-culture platform for the preclinical propagation of primary MM cells ex vivo. Cell growth was compared to yet established 2D and liquid overlay systems. MM cell lines (MMCL: RPMI-8226, U266, OPM-2) and primary patient specimens were tested. Drug sensitivity was examined by exploring the cytotoxic effect of bortezomib and the deubiquitinase inhibitor auranofin under various conditions. RESULTS: In contrast to 2D and liquid overlay, cell proliferation in the 3D array followed a sigmoidal curve characterized by an initial growth delay but more durable proliferation of MMCL over 12 days of culture. Primary MM specimens did not expand in ex vivo monoculture, but required co-culture support by a human stromal cell line (HS-5, MSP-1). HS-5 induced a \u3e fivefold increase in cluster volume and maintained long-term viability of primary MM cells for up to 21 days. Bortezomib and auranofin induced less cytotoxicity under 3D vs. 2D condition and in co- vs. monoculture, respectively. CONCLUSIONS: This study introduces a novel model that is capable of long-term propagation and drug testing of primary MM specimens ex vivo overcoming some of the pitfalls of currently available in vitro models

Soluble B-cell maturation antigen in lacrimal fluid as a potential biomarker and mediator of keratopathy in multiple myeloma

Belantamab mafodotin (belantamab) is a first-in-class anti-BCMA antibody-drug conjugate approved for the treatment of triple-class refractory multiple myeloma. It provides a unique therapeutic option for patients ineligible for CAR-T and bispecific antibody therapy, and/or patients progressing on anti-CD38 treatment where CAR-T and bispecifics might be kept in reserve. Wider use of the drug can be challenged by its distinct ocular side effect profile, including corneal microcysts and keratopathy. While dose reduction has been the most effective way to reduce these toxicities, the underlying mechanism of this BCMA off-target effect remains to be characterized. In this study, we provide the first evidence for soluble BCMA (sBCMA) in lacrimal fluid and report on its correlation with tumor burden in myeloma patients. We confirm that corneal cells do not express BCMA, and show that sBCMA-belantamab complexes may rather be internalized by corneal epithelial cells through receptor-ligand independent pinocytosis. Using an hTcEpi corneal cell-line model, we show that the pinocytosis inhibitor EIPA significantly reduces belantamab-specific cell killing. As a proof of concept, we provide detailed patient profiles demonstrating that, after belantamab-induced cell killing, sBCMA is released into circulation, followed by a delayed increase of sBCMA in the tear fluid and subsequent onset of keratopathy. Based on the proposed mechanism, pinocytosis-induced keratopathy can be prevented by lowering the entry of sBCMA into the lacrimal fluid. Future therapeutic concepts may therefore consist of belantamab-free debulking therapy prior to belantamab consolidation and/or concomitant use of gamma-secretase inhibition as currently evaluated for belantamab and nirogacestat in ongoing studies

Cabozantinib in Advanced Hepatocellular Carcinoma: Efficacy and Safety Data from an International Multicenter Real-Life Cohort

Background and Aims: The multikinase inhibitor cabozantinib has been approved for hepatocellular carcinoma (HCC) previously treated with sorafenib. We report safety and efficacy data of an international, multicenter, real-life cohort of patients with advanced HCC treated with cabozantinib. Methods: Patients with HCC who were treated with cabozantinib were retrospectively identified across 11 centers in Austria, Switzerland, and Germany. Patients’ characteristics, adverse events, duration of treatment and overall survival (OS) data were analyzed until April 1, 2020. Results: Eighty-eight patients from 11 centers were included. The predominant underlying liver diseases were NAFLD/NASH in 26 (30%) and hepatitis C infection in 21 (24%) patients. Seventy-eight patients (89%) were classified as Barcelona clinic liver cancer (BCLC) stage C. Sixty patients (68%) were Child-Pugh A, whereas 22 (25%) were Child-Pugh B, respectively. Cabozantinib was used as systemic second- and third-line or later treatment in 41 (47%) and 46 (52%) patients, respectively. The following best responses under cabozantinib were documented: partial response in 6 (7%), stable disease in 28 (32%), and progressive disease in 28 (32%) patients, respectively. Fifty-two patients (59%) died during follow-up. The median OS from start of cabozantinib treatment was 7.0 months in the entire cohort and 9.7 months in Child-Pugh A patients, while Child-Pugh B patients had a median OS of 3.4 months, respectively. Thirty-seven (42%) patients fulfilled the CELESTIAL inclusion and exclusion criteria, showing a median OS of 11.1 months. Most common adverse events were fatigue (15.6%) and diarrhea (15.6%). Conclusion: Cabozantinib treatment was effective, safe, and feasible in patients with advanced HCC in patients with compensated cirrhosis. Patients in the real-life setting had more advanced liver disease – in which 25% of patients were Child-Pugh B. However, OS in patients with Child-Pugh A cirrhosis was similar to that reported in the phase 3 trial (CELESTIAL)

Large registry analysis to accurately define second malignancy rates and risks in a well-characterized cohort of 744 consecutive multiple myeloma patients followed-up for 25 years

Additional malignancies in multiple myeloma patients after first-line and maintenance treatment have been observed, questioning whether specific risks exist. Second primary malignancies have also gained attention since randomized data showed associations to newer drugs. We have conducted this large registry analysis in 744 consecutive patients and analyzed: 1) frequency and onset of additional malignancies; and 2) second primary malignancy- and myeloma-specific risks. We assessed the frequency of additional malignancies in terms of host-, myeloma- and treatment-specific characteristics. To compare these risks, we estimated cumulative incidence rates for second malignancies and myeloma with Fine and Gray regression models taking into account competing risks. Additional malignancies were found in 118 patients: prior or synchronous malignancies in 63% and subsequent in 37%. Cumulative incidence rates for second malignancies were increased in IgG-myeloma and decreased in bortezomib-treated patients (P<0.05). Cumulative incidence rates for myeloma death were increased with higher stage and age, but decreased in IgG-subtypes and due to anti-myeloma treatment (P<0.05). Cytogenetics in patients acquiring second primary malignancies were predominantly favorable, suggesting that indolent myeloma and long disease latency may allow the manifestation of additional malignancies. An assessment of the Surveillance, Epidemiology, and End Result Program of the National Cancer Institute and our data with long-term follow up of 25 years confirmed a prevalence of second malignancy of 10% at 25 years, whereas death from myeloma decreased from 90% to 83%, respectively. Our important findings widen our knowledge of second malignancies and show that they are of increasing relevance as the prognosis in myeloma improves and mortality rates decrease

Regulatory Programs of B-cell Activation and Germinal Center Reaction Allow B-ALL Escape from CD19 CAR T-cell Therapy

Chimeric antigen receptor (CAR) T-cell therapy has led to tremendous successes in the treatment of B-cell malignancies. However, a large fraction of treated patients relapse, often with disease expressing reduced levels of the target antigen. Here, we report that exposing CD19(+) B-cell acute lymphoblastic leukemia (B-ALL) cells to CD19 CAR T cells reduced CD19 expression within hours. Initially, CD19 CAR T cells caused clustering of CD19 at the T cell-leukemia cell interface followed by CD19 internalization and decreased CD19 surface expression on the B-ALL cells. CD19 expression was then repressed by transcriptional rewiring. Using single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin using sequencing, we demonstrated that a subset of refractory CD19(low) cells sustained decreased CD19 expression through transcriptional programs of physiologic B-cell activation and germinal center reaction. Inhibiting B-cell activation programs with the Bruton's tyrosine kinase inhibitor ibrutinib increased the cytotoxicity of CD19 CAR T cells without affecting CAR T-cell viability. These results demonstrate transcriptional plasticity as an underlying mechanism of escape from CAR T cells and highlight the importance of combining CAR T-cell therapy with targeted therapies that aim to overcome this plasticity