Rėda Matuzevičienė

Scarce data exist on double maintenance in transplant-eligible high-risk (HR) newly diagnosed multiple myeloma (NDMM) patients. This prospective phase 2 study enrolled 120 transplant-eligible NDMM patients. The treatment consisted of four cycles of ixazomib–lenalidomide–dexamethasone (IRD) induction plus autologous stem cell transplantation followed by IRD consolidation and cytogenetic risk-based maintenance therapy with lenalidomide + ixazomib (IR) for HR patients and lenalidomide (R) alone for NHR patients. The main endpoint of the study was undetectable minimal residual disease (MRD) with sensitivity of −5 by flow cytometry at any time, and other endpoints were progression-free survival (PFS) and overall survival (OS). We present the preplanned analysis after the last patient has been two years on maintenance. At any time during protocol treatment, 28% (34/120) had MRD < 10⁻⁵ at least once. At two years on maintenance, 66% of the patients in the HR group and 76% in the NHR group were progression-free (p = 0.395) and 36% (43/120) were CR or better, of which 42% (18/43) had undetectable flow MRD −5. Altogether 95% of the patients with sustained MRD −5, 82% of the patients who turned MRD-positive, and 61% of those with positive MRD had no disease progression at two years on maintenance (p < 0.001). To conclude, prolonged maintenance with all-oral ixazomib plus lenalidomide might improve PFS in HR patients.

(1) Background: At diagnosis, multiplemyeloma risk estimation includes disease burden, end-organ damage, and biomarkers, with increasing emphasis on genetic abnormalities. Multicolor flow cytometry (MFC) is not always considered in risk estimation. We demonstrate associations found between genetic abnormalities and antigen expression of plasma cells measured by MFC. (2) Methods: Single nucleotide polymorphism microarray (SNP-A) karyotyping as well as MFC using standardized next-generation flow (NGF) panels and instrument settings were performed from bone marrow aspirates at the time of diagnosis. (3) Results: We uncovered specific immunophenotype features related to different genetic risk factors. Specifically, we found higher malignant/normal plasma cell ratio and lower expression of CD27, CD38, CD45, CD56, CD117 and CD138 in higher-risk genetic groups or risk categories.

Background: In this study, we investigated the yield and composition of extracellular vesicles (EVs) derived from 40- to 60-year-old healthy male controls and post-myocardial infarction (post-MI) patients' blood samples and assessed their pro-inflammatory and oxidative-related properties. Our study aimed to determine the EV yield and composition differences between both groups and to find out if there were differences between EV-mediated oxidative stress reactions. Material/Methods: Fifteen post-MI patients and 25 healthy individuals were included. EVs were isolated by ultracentrifugation and analyzed using nanotracking analysis (NTA), western blotting and fluorescent flow cytometry (FFC). Oxidative stress (OS) in blood samples was identified by measuring malondialdehyde concentration from serum, while EVs-induced OS was measured in the human vein endothelium cells (HUVEC) using H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence as a marker. Results: We found higher EVs concentration in healthy controls than in the post-MI group (7.07 +/- 3.1 E+10 ml vs 3.1 +/- 1.9 E+10 ml, P<0.001) and a higher level of CD9-positive exosomes (MFI 275 +/- 39.5 vs 252 +/- 13, P<0.001). Post-MI patients' EVs carry pro-oxidative nicotinamide adenine dinucleotide phosphate (NADPH) oxidases isoforms NOX1 (NADPH oxidase 1), NOX5 (NADPH oxidase 5) and NOX2 (NADPH oxidase 2) and anti-oxidative thioredoxin, extracellular signal-regulated kinases 1/2 (ERK1/2), and protein kinase B (Akt B). In the post-MI EVs, there was a higher predominance of enzymes with anti-oxidative effects, leading to weaker OS-inducing properties in the Conclusions: We conclude that post-MI patient blood sample EVs have stronger anti- than pro-oxidative properties and these could help fight against post-MI consequences.

Background: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression‐free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter‐laboratory variation in MM MRD reporting.Methods: Seventeen participants from 13 countries performed blinded analysis of the same eight de‐identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re‐analyzed for MRD and other bone marrow cells (Stage 2). Inter‐laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3).Results: In Stage 1, participants agreed on MRD+/MRD− status 89% and 68% of the time respectively. Inter‐observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD− status improved to 74%. Better consistency enumerating all parameters among operators resulted in near‐unanimous agreement on sample adequacy.Conclusion: Uniform flow cytometry data analysis substantially reduced inter‐laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

Higher circulating T regulatory cell (Treg) numbers have been found in untreated patients with chronic lymphocytic leukemia (CLL) compared to healthy subjects and correlated with progressive disease as well as time to first treatment in low-risk patients [1]. Some agents can reduce Treg numbers in CLL patients, but there are no data on the prognostic role of Treg dynamics and patient outcome. We present data from the LT-CLL-001 study, in which the clinical benefit of dose-dense high dose methylprednisolone (HDMP) and rituximab (Rtx) combination in relapsed or refractory high-risk patients with CLL was evaluated [2]. During the study, the change of T regulatory cell frequencies was measured in relation to overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). Twenty-nine CLL patients with clinically or biologically high-risk disease were included. Treg frequency was evaluated at screening, after three treatment courses, and at the end of therapy. Significant reduction of the median frequencies of Treg during treatment was observed: median (range) of Treg0-3 after three treatment courses was 2.14% (-1.84%- 9.42%), p < 0.001 and median (range) of Treg0-6 was 1.01% (-2.95%- 8.35%, p = 0.004). Patients with deeper Treg reduction between screening and three treatment courses had significantly better PFS and OS (Table 1 & 2). Our data for the first time show that HDMP and Rtx combination reduces Treg frequency in pretreated CLL patients. Early and deeper Treg reduction is an independent prognostic factor for longer PFS and OS. (ClinicalTrials.gov identifier: NCT005 58181).

Background and Objectives: BTK and BCL2 inhibitors have changed the treatment paradigms of high-risk and elderly patients with chronic lymphocytic leukemia (CLL), but their long-term efficacy and toxicity are still unknown and the costs are considerable. Our previous data showed that Rituximab (Rtx) and high-dose methylprednisolone (HDMP) can be an effective and safe treatment option for relapsed high-risk CLL patients. Materials and Methods: We explored the efficacy and safety of a higher Rtx dose in combination with a shorter (3-day) schedule of HDMP in relapsed elderly or unfit CLL patients. Results: Twenty-five patients were included in the phase-two, single-arm trial. The median progression free survival (PFS) was 11 months (range 10–12). Median OS was 68 (range 47–89) months. Adverse events (AE) were mainly grade I–II° (77%) and no deaths occurred during the treatment period. Conclusions: 3-day HDMP and Rtx was associated with clinically meaningful improvement in most patients. The median PFS in 3-day and 5-day HDMP studies was similar and the toxicity of the 3-day HDMP schedule proved to be lower. The HDMP and Rtx combination can still be applied in some relapsed high-risk and elderly or unfit CLL patients if new targeted therapies are contraindicated or unavailable. (ClinicalTrials.gov identifier: NCT01576588).

(2018). ETV6 and NOTCH1 germline variants in adult acute leukemia. Leukemia & Lymphoma: Vol. 59, No. 4, pp. 1022-1024.

Aim: To evaluate quantitative changes in B, NK and T lymphocyte subsets in peripheral blood of children with acute lymphoblastic leukemia (ALL) undergoing chemotherapy. Patients and Methods: Children with ALL were treated according to NOPHO ALL 2008 protocol. Levels of B lymphocytes (CD19⁺), NK cells (CD3⁻CD56⁺) and subsets of T lymphocytes (CD3⁺CD4⁺, CD4⁺CD25⁺Foxp3⁺, CD3⁺CD8⁺, CD3⁺CD8⁺CD57⁺, CD3⁺CD8⁺CD57⁻) in peripheral blood were analyzed by flow cytometry prior and during treatment with cytotoxic drugs. Results: Immunological analyses were performed in 25 children with ALL. Levels of B and NK lymphocytes decreased continuously during chemotherapy. In contrast, levels of most T lymphocyte subsets decreased only transiently and returned to pretreatment levels by days 78 to 85. The only T lymphocyte subset that did not return to the pretreatment level contained senescent CD3⁺CD8⁺CD57⁺ lymphocytes. Conclusion: Immunomodulating action of chemotherapy in children with ALL results in reduction of proportion of senescent CD8⁺ T lymphocytes.