Spatial transcriptomics reveals profound subclonal heterogeneity and T-cell dysfunction in extramedullary myeloma.

Extramedullary disease (EMD) is a high-risk feature of multiple myeloma (MM) and remains a poor prognostic factor even in the era of novel immunotherapies. Here we applied spatial transcriptomics (tomo-seq [n=2] and 10X Visium [n=12]), and single-cell RNA sequencing (scRNAseq [n=3]) to a set of 14 EMD biopsies to dissect the three-dimensional architecture of tumor cells and their microenvironment. Overall, the infiltrating immune and stromal cells showed both intra- and inter-patient variation with no uniform distribution over the lesion. We observed substantial heterogeneity at the copy number level within plasma cells, including the emergence of new subclones in circumscribed areas of the tumor, consistent with genomic instability. We further identified spatial expression differences of GPRC5D and TNFRSF17, two important antigens for bispecific antibody therapy. EMD masses were infiltrated by various immune cells, including T-cells. Notably, exhausted TIM3+/PD-1+ T-cells diffusely co-localized with MM cells, whereas functional and activated CD8+ T-cells showed a focal infiltration pattern along with M1 macrophages in otherwise tumor-free regions. This segregation of fit and exhausted T-cells was resolved in the case of response to T-cell engaging bispecific antibodies. MM cells and microenvironment cells were embedded in a complex network that influenced immune activation and angiogenesis, and oxidative phosphorylation represented the major metabolic program within EMD lesions. In summary, spatial transcriptomics has revealed a multicellular ecosystem in EMD with checkpoint inhibition and dual targeting as potential new therapeutic avenues.

Changes in T-cell subsets, preexisting cytopenias and hyperferritinaemia correlate with cytopenias after BCMA targeted CAR T-cell therapy in relapsed/refractory multiple myeloma: Results from a prospective comprehensive biomarker study.

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.

High-dose carfilzomib achieves superior anti-tumor activity over low-dose and recaptures response in relapsed/refractory multiple myeloma resistant to lowdose carfilzomib by co-inhibiting the ß2 and ß1 subunits of the proteasome complex.

Optimal carfilzomib dosing is a matter of debate. We analyzed the inhibition profiles of proteolytic proteasome subunits ß5, ß2 and ß1 after low-dose (20/27 mg/m2) versus high-dose (≥36 mg/m2) carfilzomib in 103 pairs of peripheral blood mononuclear cells from patients with relapsed/refractory (RR) multiple myeloma (MM). ß5 activity was inhibited (median inhibition >50%) in vivo by 20 mg/m2, whereas ß2 and ß1 were co-inhibited only by 36 and 56 mg/m2, respectively. Coinhibition of ß2 (P=0.0001) and ß1 activity (P=0.0005) differed significantly between high-dose and low-dose carfilzomib. Subsequently, high-dose carfilzomib showed significantly more effective proteasome inhibition than low-dose carfilzomib in vivo (P=0.0003). We investigated the clinical data of 114 patients treated with carfilzomib combinations. High-dose carfilzomib demonstrated a higher overall response rate (P=0.03) and longer progression-free survival (PFS) (P=0.007) than low-dose carfilzomib. Therefore, we escalated the carfilzomib dose to ≥36 mg/m2 in 16 patients who progressed during low-dose carfilzomib-containing therapies. High-dose carfilzomib recaptured response (≥ partial remission) in nine (56%) patients with a median PFS of 4.4 months. Altogether, we provide the first in vivo evidence in RRMM patients that the molecular activity of high-dose carfilzomib differs from that of low-dose carfilzomib by coinhibition of ß2 and ß1 proteasome subunits and, consequently, high-dose carfilzomib achieves a superior anti-MM effect than low-dose carfilzomib and recaptures the response in RRMM resistant to low-dose carfilzomib. The optimal carfilzomib dose should be ≥36 mg/m2 to reach a sufficient anti-tumor activity, while the balance between efficacy and tolerability should be considered in each patient.

Salvage therapy with "Dara-KDT-P(A)CE" in heavily pretreated, high-risk, proliferative, relapsed/refractory multiple myeloma.

The multi-agent therapy "VDT-PACE" represents an established regimen in relapsed/refractory multiple myeloma (RRMM). Here, we report on our experience with a "modified VDT-PACE" incorporating new generation anti-MM agents daratumumab and carfilzomib ("Dara-KDT-P(A)CE"). We retrospectively analyzed 38 patients with RRMM treated with "Dara-KDT-P(A)CE". The median age was 62 (range 45-82) years, and the patients were heavily pretreated with a median of 5 (range 2-12) prior lines of therapy. Twenty-one (55%) patients suffered from penta-refractory MM. High-risk cytogenetics was present in 31 (81%) patients. The patients received a median of 2 (range 1-10) cycles of this therapy, and the overall response rate (ORR) was 70%. Patients with penta-refractory MM and high-risk cytogenetics showed similar ORR of 65% and 79%, respectively. The median progression-free survival (PFS) and overall survival were 4.1 (95% CI 2.7-5.4) and 8.4 (95% CI 6.7-10.0) months, respectively. Patients with lactate dehydrogenase >250 IU/L showed significantly shorter PFS in comparison with others patients (p = 0.006). We used this regimen as bridging therapy prior to chimeric antigen receptor T-cell infusion in four patients. In conclusion, "Dara-KDT-P(A)CE" is an effective salvage therapy for patients with heavily pretreated, multi-refractory, high-risk RRMM lacking alternative options.

Obinutuzumab and venetoclax induced complete remission in a patient with ibrutinib-resistant non-nodal leukemic mantle cell lymphoma.

We herein report the case of a 73-year-old male patient who was diagnosed with leukemic non-nodal MCL. This patient had received six cycles of bendamustine, which resulted in a transient remission, and a second-line therapy with ibrutinib, which unfortunately failed to induce remission. We started a treatment with single-agent obinutuzumab at a dose of 20 mg on day 1, 50 mg on day 2-4, 330 mg on day 5, and 1000 mg on day 6. The laboratory analysis showed a rapid decrease of leukocyte count. Four weeks later, we repeated the treatment with obinutuzumab at a dose of 1000 mg q4w and started a therapy with venetoclax at a dose of 400 mg qd, which could be increased to 800 mg qd from the third cycle. This combination therapy was well tolerated. The patient achieved a complete remission (CR) after three cycles of obinutuzumab and venetoclax. To date, the patient has a progression-free survival of 17 months under ongoing obinutuzumab maintenance q4w. This is the first report about obinutuzumab and venetoclax induced CR in rituximab-intolerant patient with an ibrutinib-resistant MCL. This case suggests that obinutuzumab- and venetoclax-based combination therapy might be salvage therapy in patients with ibrutinib-resistant MCL.

Liver stiffness as a prognostic parameter and tool for risk stratification in advanced cardiac transthyretin amyloidosis.

BACKGROUND: In light of increasing therapeutic options, risk stratification of advanced cardiac transthyretin amyloidosis (ATTR-CA) is gaining clinical importance to avoid ineffective treatments. Liver stiffness as a marker of hypervolemia and hepatic congestion might predict mortality in advanced ATTR-CA and allow to identify patients at highest risk. METHODS: Proven ATTR-CA patients underwent repeated vibration-controlled transient elastography (VTCE) and standardized serial workup within the local amyloidosis cohort study AmyKoS. Spearman correlation analyses and Cox regressions were performed to evaluate the prognostic value. RESULTS: 41 patients with ATTR-CA were included with median age of 76.6 (55.1-89.1) years, of which 90.2% were male and > 92% wild-type ATTR-CA. In total, 85 VCTE examinations were performed. Median follow-up was 43.7 (2.4-75.6) months; 26.8% of the patients died. At the first clinical evaluation, median left ventricular (LV) absolute global longitudinal strain (GLS) was 11.4 (5.2-19.0) % and median liver stiffness was 6.3 (2.4-22.9) kPa, both significantly correlated with mortality. NT-proBNP possessed statistically significant predictive power in ATTR-CA with more preserved LV function (absolute GLS ≥ 10), whereas stiffness seemed to be more discriminative for ATTR-CA with absolute GLS < 10. The use of alternative congestion surrogates such as liver vein dilation and tricuspid regurgitation peak velocity (tr-vmax) showed congruent results. CONCLUSION: Liver stiffness shows prognostic value regarding all-cause mortality and allows risk stratification in advanced ATTR-CA, particularly in those with markedly impaired longitudinal LV function. These results are transferable to other congestion surrogates.

Incremental prognostic utility of congestion markers in cardiac transthyretin amyloidosis.

BACKGROUND/AIMS: Congestion is prognostically relevant in cardiac transthyretin amyloidosis (ATTR-CA), but whether congestion has an incremental prognostic value beyond the well-established, congestion-sensitive NT-proBNP is unknown. Therefore, we aimed to comparatively evaluate the prognostic utility of several congestion surrogates over NT-proBNP. METHODS: We estimated hazard ratios by Cox proportional hazards regressions with time-varying covariates from a panel data set of the local amyloidosis cohort study AmyKoS. Different models were compared by using chi(χ)2-statistics measuring overall model significance. RESULTS/CONCLUSION: 131 ATTR-CA patients (wild-type 84.0%, hereditary 6.9%, without genetic testing 9.2%; median age 78.7 (quartiles 73.3, 82.1) years; 85.5% male) with 566 observations across a median follow-up of 38.2 (30.6; 48.2) months were analyzed. 83.2% received disease-modifying treatment; 20.6% participated concurrently in placebo-controlled gene silencer trials. Information on congestion improved biomarker-driven risk stratification and identified patients at the highest risk. Echocardiographic congestion markers performed better than clinical findings and daily diuretic use/dosage. Relevant adjusters were daily diuretic dosage, disease-modifying treatment, eGFR, and right atrial volume. NT-proBNP and the tricuspid regurgitation peak velocity (tr-vmax) provided an easy-to-use stratification with overall model performance similar to NAC and Mayo staging systems. Further analyses are necessary for validation and to identify the optimal cut points of the congestion markers.

Carfilzomib Based Treatment Strategies in the Management of Relapsed/Refractory Multiple Myeloma with Extramedullary Disease.

Published experience with carfilzomib in patients with relapsed/refractory multiple myeloma (RRMM) and extramedullary disease (EMD) is still limited. The current study aimed to assess the efficacy and safety of carfilzomib containing therapy regimens in EMD. We retrospectively analyzed 45 patients with extramedullary RRMM treated with carfilzomib from June 2013 to September 2019. The median age at the start of carfilzomib was 64 (range 40-80) years. Twenty (44%) and 25 (56%) patients had paraosseous manifestation and EMD without adjacency to bone, respectively. The serological overall response rate (ORR) was 59%. Extramedullary response was evaluable in 33 patients, nine (27%) of them achieved partial remission (PR) (ORR = 27%). In 15 (33%) patients, we observed no extramedullary response despite serological response. The median progression-free survival (PFS) and overall survival (OS) were five (95% CI, 3.5-6.5) and ten (95% CI, 7.5-12.5) months, respectively. EMD without adjacency to bone was associated with a significantly inferior PFS (p = 0.004) and OS (p = 0.04) compared to paraosseous lesions. Carfilzomib based treatment strategies showed some efficacy in heavily pretreated patients with extramedullary RRMM but could not overcome the negative prognostic value of EMD. Due to the discrepancy between serological and extramedullary response, evaluation of extramedullary response using imaging is mandatory in these patients.

Sequential CD38 monoclonal antibody retreatment leads to deep remission in a patient with relapsed/refractory multiple myeloma.

We report on a currently 76-year-old female patient with relapsed/refractory (RR) multiple myeloma (MM) treated at our institution. This patient had received six lines of therapy including tandem autologous stem cell transplant, proteasome inhibitor, immunomodulatory drugs and CD38 antibody MOR202. At the last relapse, she progressed during treatment with pomalidomide and MOR202. In an individualized therapy concept, we started a multi-agent salvage therapy with pomalidomide, bortezomib, doxorubicin, dexamethasone, and CD38 antibody daratumumab ("Pom-PAD-Dara"), which resulted in a stringent complete remission with minimal residual disease (MRD) negativity after nine cycles. So far, our patient shows a progression free survival of more than 12 months. Our case demonstrates the feasibility of successful CD38 antibody retreatment in a patient with heavily pretreated CD38 antibody resistant MM.