Impact of the NK Cell Receptor LIR-1 (ILT-2/CD85j/LILRB1) on Cytotoxicity against Multiple Myeloma

The role of different receptors in natural-killer- (NK-) cell-mediated cytotoxicity against multiple myeloma (MM) cells is unknown. We investigated if an enhancement of NK-cell-mediated cytotoxicity against MM could be reached by blocking of the inhibitory leukocyte immunoglobulin-like receptor 1 (LIR-1). Our investigations revealed high levels of LIR-1 expression not only on the NK cell line NK-92, but also on myeloma cells (MOLP-8, RPMI8226) as well as on a lymphoblastoid cell line (LBCL; IM-9). Subsequent cytotoxicity assays were designed to show the isolated effects of LIR-1 blocking on either the effector or the tumor side to rule out receptor-receptor interactions. Although NK-92 was shown to be capable of myeloma cell lysis, inhibition of LIR-1 on NK-92 did not enhance cytotoxicity. Targeting the receptor on MM and LBCL did not also alter NK-92-mediated lysis. We come to the conclusion that LIR-1 alone does not directly influence NK-cell-mediated cytotoxicity against myeloma. To our knowledge, this work provides the first investigation of the inhibitory capability of LIR-1 in NK-92-mediated cytotoxicity against MM and the first functional evaluation of LIR-1 on MM and LBCL

Use of ropeginterferon in inducing graft versus myelofibrosis effect in post‐transplant myelofibrosis relapse

Key Clinical Message Here, we describe a patient with post‐transplant myelofibrosis with chronic graft‐versus‐host disease (GVHD), who showed successful molecular remission with ropeginterferon with 100% donor chimerism without any flare up of GVHD. He was initially diagnosed with polycythemia vera (PV) which progressed to myelofibrosis after 6 years. The MYSEC (Myelofibrosis Secondary to PV and ET‐Prognostic Model) and MTSS (myelofibrosis transplant scoring system) scores were 13.1 and 4, respectively, and the patient was in intermediate risk group. He underwent an allogenic stem cell transplant; however, his disease gradually progressed and was administered two donor lymphocyte infusions with minimal response. A second allogeneic transplant was performed, which led to a persistent molecular remission for more than a decade, although he developed chronic skin graft‐versus‐host disease (GVHD). The JAK2 V617F levels started to increase 10 years post‐transplant with ongoing chronic GVHD and a corresponding decrease in donor chimerism levels. He was administered ropeginterferon, which led to a decrease in JAK 2617F to undetectable levels. A graft versus myelofibrosis effect was attained with reversal to 100% donor chimerism, and he has since maintained a molecular remission with undetectable JAK 2617F levels. Chronic GVHD made him ineligible for donor lymphocyte infusions later. Thus, ropeginterferon was successful in inducing graft versus myelofibrosis effect, leading to a molecular response with no flare up of GVHD. The use of ropeginterferon needs to be further evaluated in larger cohorts of post‐transplant myelofibrosis patients

Role of pre-transplant MRD level detected by flow cytometry in recipients of allogeneic stem cell transplantation with AML.

OBJECTIVES AND METHODS We analyzed the impact of pre-transplant MRD level in bone marrow measured by flow cytometry using "different from normal" method on outcomes for 189 AML patients (108 males; median age, 58 (21-80) years). All patients were subdivided into negative (n=96), "low" (0.1-0.5%, n=32) and "high" MRD (>0.5%, n=61) groups. RESULTS In multivariate analysis, the hazard ratios for "high" and "low" MRD levels related to MRD negativity were 7.9 (95% CI 3.5-18.1, p<0.001) and 5.4 (95% CI 2.1-14, p=0.0058) for relapse; 2.3 (95% CI 1.3-4.1, p=0.006) and 1.6 (95% CI 0.82-3.3, p=0.16) for OS; 2.8 (95% CI 1.7-4.7, p<0.001) and 2.2 (95% CI 1.1-4.2, p=0.02) for LFS, respectively. We found no significant impact of "low" MRD level on relapses (0.68, 95% CI 0.33-1.4, p=0.30), OS (0.72, 95% CI: 0.36-1.5, p=0.36) and LFS (0.79, 95% CI: 0.42-1.5, p=0.46) related to "high" MRD group. CONCLUSIONS Presence of detectable MRD was indicative for a high relapse risk, low LFS and OS. "Low" MRD level showed no significant impact on relapse, LFS and OS related to "high" MRD group

Donor Lymphocyte Infusion and Molecular Monitoring for Relapsed Myelofibrosis After Hematopoietic Cell Transplantation

Hematopoietic cell transplantation (HCT) is a curative approach for myelofibrosis patients, but relapse is a major cause of treatment failure. We investigated the effect of donor lymphocyte infusion (DLI) in 37 patients with molecular (n = 17) or hematological relapse (n = 20) after HCT. Patients received median of 2 (range, 1–5) cumulative DLI (total of 91 infusions). Median starting dose was 1 × 106 cells/kg, escalated by half-log ≥6 weeks if no response nor graft-versus-host disease (GvHD) occurred. Median time to first DLI was 40 weeks for molecular relapse versus 145 weeks for hematological relapse. Overall molecular complete response (mCR) at any time was 73% (n = 27) and was significantly higher for initial molecular relapse (88%) versus hematological relapse (60%; P = 0.05). The 6-year overall survival was 77% versus 32% (P = 0.03). Acute GvHD 2–4 occurred in 22% and half of the patients achieved mCR without any GvHD. All patients who relapsed from mCR achieved after first DLI could be salvaged with subsequent DLI, showing long-term survival. No second HCT was needed for molecular relapse versus 6 for hematological relapse. This comprehensive and largest study to date suggests molecular monitoring together with DLI as standard of care and a crucial approach to achieve excellent outcomes in relapsed myelofibrosis

5-Azacytidine Promotes an Inhibitory T-Cell Phenotype and Impairs Immune Mediated Antileukemic Activity

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ+ T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza

Post-transplant day +100 MRD detection rather than mixed chimerism predicts relapses after allo-SCT for intermediate risk AML patients transplanted in CR.

BACKGROUND Chimerism and minimal residual disease (MRD) are suggested to be prognostic for post-transplant relapses in AML patients. Nevertheless, the predictive values of both approaches in homogeneous population remain underinvestigated. Here, we suggest that MRD may have a higher predictive value for relapses than mixed chimerism (MC) in intermediate risk AML patients. PATIENTS AND METHODS 79 patients with intermediate risk AML (male, n=40, median age, 57 (19-77)) were included. MRD detection on day +100 was performed in bone marrow (multiparameter flow cytometry and quantitative real-time PCR for NPM1-mutated patients). Chimerism analysis was measured in peripheral blood. MC was defined as persistence of <99.9% of donor alleles. RESULTS The area under the ROC curve was highest for qPCR-MRD (0.93) followed by MFC-MRD (0.80) and MC (0.65). The highest relapses at 3 years were observed in day +100 qPCR-MRD positive patients (100%) followed by MFC-MRD positive patients (55%, p<0.001). No patients with MC and without detectable MRD developed relapses. The 3-year OS and LFS for patients with MC without detectable MRD were both 86% (61-96%) compared with day +100 MFC-MRD positive (OS: 61%, 36-84%; LFS: 30%, 11-59%) and with day +100 qPCR-MRD positive patients (OS: 17%, 3-56%, p=0.001; LFS: 0%, p<0.001). CONCLUSIONS In intermediate-risk AML, the qPCR-MRD on day +100 is highly predictive for relapse and long-term survival after allo-SCT, closely followed by MFC-MRD. In contrast, the chimerism status has limited predictive potential. Thus, molecular and flow-cytometric MRD monitoring in the first months post-transplant rather than MC is able to identify patients with an increased relapse risk who may benefit from early post-transplant pre-emptive intervention

Population Pharmacokinetics of Busulfan and Its Metabolite Sulfolane in Patients with Myelofibrosis Undergoing Hematopoietic Stem Cell Transplantation

For patients with myelofibrosis, allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the only curative treatment to date. Busulfan-based conditioning regimens are commonly used, although high inter-individual variability (IIV) in busulfan drug exposure makes individual dose selection challenging. Since data regarding the IIV in patients with myelofibrosis are sparse, this study aimed to develop a population pharmacokinetic (PopPK) model of busulfan and its metabolite sulfolane in patients with myelofibrosis. The influence of patient-specific covariates on the pharmacokinetics of drug and metabolite was assessed using non-linear mixed effects modeling in NONMEM®. We obtained 523 plasma concentrations of busulfan and its metabolite sulfolane from 37 patients with myelofibrosis. The final model showed a population clearance (CL) and volume of distribution (Vd) of 0.217 L/h/kg and 0.82 L/kg for busulfan and 0.021 L/h/kg and 0.65 L/kg for its metabolite. Total body weight (TBW) and a single-nucleotide polymorphism of glutathione-S-transferase A1 (GSTA1 SNP) displayed a significant impact on volume of distribution and metabolite clearance, respectively. This is the first PopPK-model developed to describe busulfan’s pharmacokinetics in patients with myelofibrosis. Incorporating its metabolite sulfolane into the model not only allowed the characterization of the covariate relationship between GSTA1 and the clearance of the metabolite but also improved the understanding of busulfan’s metabolic pathway

Accurate In-Vivo Quantification of CD19 CAR-T Cells after Treatment with Axicabtagene Ciloleucel (Axi-Cel) and Tisagenlecleucel (Tisa-Cel) Using Digital PCR

Immunotherapy with CD19-specific chimeric antigen receptor (CAR-) T cells has shown excellent efficacy in relapsed/refractory B-cell cancers. The in vivo expansion and persistence of CAR-T cells after infusion are important response- and toxicity-determining variables, but diagnostic tools are largely missing. We showed previously for axi-cel that digital PCR (dPCR) is excellently suited to monitoring CAR-T cells in vivo. Here, we aimed to develop an analogous dPCR assay for tisa-cel. To do so, we cloned and sequenced the CAR construct from the lentiviral tisa-cel vector and designed primers and Black hole quencher (BHQ) probes complimentary to sequences present in the FMC63 scFv part of axi-cel (assay A), tisa-cel (T), and both constructs (U = &ldquo;universal&rdquo;). In conjunction with excellent specificity, all assays have a detection limit of one single CAR copy, corresponding to a sensitivity of approximately 1 in 5000 cells (0.02%) for 100 ng genomic DNA (for one vector copy per transduced cell). The new universal assay was first validated using patient samples previously quantified with the axi-cel-specific dPCR and thereafter applied to quantify and monitor adoptively transferred axi-cel and tisa-cel T cells in post-infusion samples (peripheral blood, bone marrow, liquor, and ascites). Actual CAR-T counts per &micro;l were calculated, taking into account vector copy and peripheral blood mononuclear cell (PBMC) numbers, and showed very good correlation with flow cytometry results. We conclude that our novel dPCR assay is optimally suited to monitoring tisa-cel and axi-cel CAR-T cells in real-time in various body fluids

Enhanced immune reconstitution of γδ T cells after allograft overcomes negative impact of pre-transplant MRD positive status in AML patients.: γδ T cells and MRD+ AML.

BACKGROUND Minimal residual disease (MRD) prior to allogeneic stem cell transplantation (allo-SCT) in AML is a poor risk factor for outcome. The γδ T cells represents a unique minority lymphocyte population which is preferentially located in peripheral tissues, can recognize antigens in non-MHC restricted manner and plays a "bridging" role between innate and adaptive immune system. OBJECTIVES In this study, we investigated a potential graft-vs-leukaemia effect of γδ T cells reconstitution post-transplant in AML patients with pre-transplant positive minimal/measurable disease status (MRD+). STUDY DESIGN We investigated a potential graft-vs-leukaemia effect of γδ T cells reconstitution post-transplant in AML patients with pre-transplant positive MRD+. MRD assessment was performed in 202 patients (MRD+, n=100) with multicolored flow cytometry ("different from normal" strategy). Analysis for absolute concentrations of CD3+, CD4+, CD8+, NK, and γδ T cells were performed by flow cytometry according to an internal protocol at day +30 and +100 post-transplant. Differences between categorical and continuous variables were determined by Chi-square and Student's T-test, respectively. The Mann-Whitney test was used to compare medians of continuous variables. Spearman correlation was used for nonparametric assessment of correlation between different cell subsets during immune reconstitution. Kaplan Meier survival analysis and Cox regression analysis were used to investigate the associations between immune reconstitution and survival outcomes. Grays' analysis was used to compute incidences of relapses, non-relapse mortality (NRM) and graft-vs-host disease (GvHD). RESULTS Follow-up for survivors was 28 months (3-59). Younger age (≤58) of recipient and donor (<30), sex mismatch, matched donors, CMV reactivation and ATG were associated with a faster γδ T cell reconstitution. In multivariable analysis for MRD+ patients, higher than median level of γδ T cells on days +30 and +100 resulted in a significant improved leukaemia-free (HR 0.42, p=0.007 and HR 0.42, p=0.011, respectively) and overall survival (HR 0.44, p=0.038 and HR 0.33, p=0.009, respectively). Further, higher γδ T cell level on day +30 led to significant reduced risk of relapse (HR 0.36, p=0.019). No impact of γδ T cell level on day +30 and +100 could be seen in MRD- patients and no correlation with occurrence of graft-versus-host disease could be observed. CONCLUSION An enhanced immune reconstitution of γδ T cells post-transplant may overcome the higher relapse risk of pre-transplant MRD+ patients with AML