Marrow Mononuclear Cells Research Articles

Advanced Analysis and Validation of a microRNA Signature for Fanconi Anemia.

Some years ago, we reported the generation of a Fanconi anemia (FA) microRNA signature. This study aims to develop an analytical strategy to select a smaller and more reliable set of molecules that could be tested for potential benefits for the FA phenotype, elucidate its biochemical and molecular mechanisms, address experimental activity, and evaluate its possible impact on FA therapy. In silico analyses of the data obtained in the original study were thoroughly processed and anenrichment analysis was employed to identify the classes of genes that are over-represented in the FA-miRNA population under study. Primary bone marrow mononuclear cells (MNCs) from sixFA patients and sixhealthy donors as control samples were employed in the study. RNAs containing the small RNA fractions were reverse-transcribed and real-time PCR was performed in triplicate using the specific primers. Experiments were performed in triplicate.The in-silico analysis reported six miRNAs as likely contributors to the complex pathological spectrum of FA. Among these, three miRNAs were validated by real-time PCR. Primary bone marrow mononuclear cells (MNCs) reported a significant reduction in the expression level of miRNA-1246 and miRNA-206 in the FA samples in comparison to controls.This study highlights several biochemical pathways as culprits in the phenotypic manifestations and the pathophysiological mechanisms acting in FA. A relatively low number of miRNAs appear involved in all these different phenotypes, demonstrating the extreme plasticity of the gene expression modulation. This study further highlights miR-206 as a pivotal player in regulatory functions and signaling in the bone marrow mesenchymal stem cell (BMSC) process in FA. Due to this evidence, the activity of miR-206 in FA deserves specific experimental scrutiny. The results, here presented, might be relevant in the management of FA.

Mesenchymal stem cell-derived exosomes can alleviate GVHD and preserve the GVL effect in allogeneic stem cell transplantation animal models.

Mesenchymal stem cells (MSCs) can alleviate graft-versus-host disease (GVHD) in hematopoietic stem cell transplantation (HSCT). MSCs-derived exosomes (MEXs) can mirror the biological function of their parent cells. Whether MEXs can alleviate GVHD like their parent cells or not is unclear. In this study, we investigate the effects of MEXs on GVHD and graft-versus-leukemia (GVL) effect in vitro and in HSCT animal models. MSCs were produced using bone marrow mononuclear cells (MNCs), and MEXs were separated from the supernatants of MSCs. Electron microscopy, western blot, and nanoparticle tracking analysis (NTA) were used to determine the characteristics of MEXs. The immunomodulatory function of MEXs and their effects on GVHD and GVL were examined in vitro and in vivo. Like other cell-type derived exosomes, our data revealed that MEXs were also disc-shaped vesicles with a diameter of 100-200 nm under electron microscopy and were positive for the exosomal hallmark proteins. MEXs can notably inhibit the expression of costimulatory molecules and functional cytokine secretion of dendritic cells (DCs). Meanwhile, MEXs can exert suppressive effects on T lymphocyte proliferation and activation. Moreover, MEXs can also encourage the polarization of macrophages toward the M2 type. In animal HSCT models, MEXs can promote the differentiation of Treg cells in spleens, decrease the GVHD score, increase the survival rate of mice, and preserve the cytotoxic antileukemia effects of CD8+ T lymphocytes from recipient mice. These findings showed that MEXs exert their effects by inhibiting the immunomodulatory function of DCs, macrophages, and T lymphocytes. In the animal model, MEXs ameliorate the clinical symptoms of GVHD, while maintaining the antitumor effects of CD8+ T lymphocytes. Therefore, it can be inferred that MEXs can separate GVHD from GVL in HSCT. Our study suggests that MEXs have broad clinical application potential in the prevention and treatment of GVHD in HSCT in the near future.

Bone Marrow Interstitial Light-Chain Amyloid and Its Microenvironment

Amyloid deposition and neighboring tissue responses remain poorly understood. Patient series have shown that with Congo red staining of marrow biopsies about 20% of AL patients have interstitial deposits, 40% have vascular and 40% have no amyloid seen and that marrow amyloid is rare in patients with myeloma. (Ann Hematol. 2011;90(1):101-6; Am J Clin Pathol. 2003;120(4):610-6) The constituents of the deposits have been summarized for 65 AL biopsies, a third with extensive interstitial deposits; apolipoproteins E, A-IV and J are prominently present in the examples offered as well as in an extensive summary of the amyloid proteome.(Amyloid. 2022;29(3):156-64; Crit Rev Biochem Mol Biol. 2021;56(5):526-42) With the goal of achieving a better understanding of the impact AL amyloid deposits might have on the marrow microenvironment we undertook a systematic assessment, within the limitations of specimen availability and quality, of the CD138-depleted marrow mononuclear cells (MNC) and marrow and blood plasma from AL patients with (+MA) and without (-MA) interstitial marrow amyloid. Data on the 49 consenting AL patients are archived in the Harvard Dataverse (https://doi.org/10.7910/DVN/TDFMAI). We compared +MA and -MA CD138-depleted MNC from AL patients by gene expression profiling (GEP) on the Illumina platform; reads were mapped to the UCSC hg19 human genome with Tophat 2/Bowtie 2. Normalization and differential expression analyses were performed with Cuffdiff.(Methods Mol Biol. 2016;1374:339-61) We also compared a different set of +MA and -MA CD138-depleted MNC by single-cell RNA-sequencing (scRNA seq) employing the 10X Genomics Chromium Single Cell 3′ method in our core facility and processing the samples for evaluation through CellRanger, Seurat v. 4.0.3 and the Azimuth marrow reference-based method at the 31 cell type resolution. In addition, we compared peripheral blood and marrow plasma from these patients by immunoprecipitation and immunoblot with respect to apolipoproteins and free light chains (FLC) in complex with the erythroid protein TMCC2 and by ELISA for marrow apolipoprotein E and J levels. GEP showed no differential expression of genes for proteolytic enzymes or apolipoproteins between the groups but +MA cases had significantly up-regulated erythroid genes involved in oxygen transport, including transmembrane and coiled-coil domain family 2 ( TMCC2). In +MA marrows at the single-cell level, CD14+ monocytes were increased by 22%, granulocyte-monocyte progenitors decreased by 66%, and erythroid-megakaryocyte progenitors and early and late erythroid progenitors increased up to five-fold. Gene enrichment studies showed that in +MA marrows pathways for TNFα signaling via NFκ-B were significantly active (Figure 1); immune activation and monocyte hypoxia were also significantly enriched. As shown in Figure 2, apolipoprotein J is strongly associated with FLC in blood and E is not, while in +MA marrow plasma apolipoproteins J and E are strongly present in association with FLC. By ELISA, there is also significantly more apolipoprotein E and J in +MA marrow plasma. Our data suggest that erythroid-related activity is enhanced in marrows with interstitial amyloid and that a red blood cell related protein, TMCC2, is associated with apolipoproteins and FLC, possibly providing a loose cargo of chaperones that may abet fibril formation by FLC with predisposed secondary structures whose fragmentation sites are made optimally accessible by the loose complex; sites in the FR4 and 5' constant region of AL FLC may be highly vulnerable if exposed in such fashion to perivisceral proteases associated with matrix remodeling. The marrow microenvironment is clearly impacted by the presence of interstitial amyloid. In HSC from +MA marrows there is increased signaling in pathways that affect lineage commitments such as TNF-α/NF-κB and PI3 kinase/AKT/mTOR. In monocytes from +MA specimens there is evidence of modulation of NF-kB. Both alterations are possibly due to apolipoprotein E, although there is no evidence that monocyte transcription of APO E in +MA CD138-depleted marrow MNC is increased. In conclusion, marrows with interstitial amyloid provide opportunities to study amyloid's impact on cellular behavior, regenerative activity and chaperone involvement in amyloid deposition.

Disturbed Cytokine Profile in Adjuvant Arthritis – A Target of the Therapeutic Potential of Dendritic Cells Derived From Cryopreserved Precursors

Background. One of the primary causes of rheumatoid arthritis development is the disruption of the immune system's natural tolerance to its own antigens, leading to an imbalance in the body's cytokine profile. A promising method of correcting such a condition is restoring antigen-specific tolerance, in the formation of which tolerogenic dendritic cells (tolDCs) take part. Objective. Experimental substantiation of the possibility of correcting the cytokine profile of animals with adjuvant arthritis (AA) by using tolDCs from cryopreserved bone marrow precursors. Methods. The study was carried out on the CBA/H mice. The development of AA was assessed using a clinical indicator – the arthritis index. The levels of pro- (TNF-a, IL-6, IFN-g) and anti-inflammatory (IL-10, IL-4) cytokines in the blood serum of AA-afflicted animals were measured before and after administration of tolDCs. These tolDCs were obtained from native (NatDCs) or cryopreserved (CryoDCs) using different methods bone marrow mononuclear cells (MNCs). On the 14th day after inducing AA, the animals received intravenous injections of tolDCs (5´105/mouse). One week later, the cytokine levels in the animals' blood serum and the arthritis index were assessed. Results. Throughout the development of AA, a unidirectional increase in TNF-α and IL-6 levels and a reduction in the content of anti-inflammatory cytokines were observed, which was accompanied by joint swelling in the animals. CryoDCs exhibited a more pronounced corrective effect on both pro- and anti-inflammatory cytokines compared to NatDCs, as evidenced by a decrease in the arthritis index, a clinical manifestation of the pathology. Conclusions. The possibility of correcting the disturbed cytokine profile and the clinical state of animals during the development of AA through the use of tolDCs derived from cryopreserved MNCs has been proven. Specific cryopreservation conditions for MNCs have been developed, which facilitate the generation of tolDCs from them with a greater capacity, compared to derivatives of native MNCs, to correct the cytokine profile and clinical status of animals with AA.

Optimizing the Seeding Density of Human Mononuclear Cells to Improve the Purity of Highly Proliferative Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) hold considerable promise for regenerative medicine. Optimization of the seeding density of mononuclear cells (MNCs) improves the proliferative and differentiation potential of isolated MSCs. However, the underlying mechanism is unclear. We cultured human bone marrow MNCs at various seeding densities (4.0 × 104, 1.25 × 105, 2.5 × 105, 6.0 × 105, 1.25 × 106 cells/cm2) and examined MSC colony formation. At lower seeding densities (4.0 × 104, 1.25 × 105 cells/cm2), colonies varied in diameter and density, from dense to sparse. In these colonies, the proportion of highly proliferative MSCs increased over time. In contrast, lower proliferative MSCs enlarged more rapidly. Senescent cells were removed using a short detachment treatment. We found that these mechanisms increase the purity of highly proliferative MSCs. Thereafter, we compared MSCs isolated under optimized conditions with a higher density (1.25 × 106 cells/cm2). MSCs under optimized conditions exhibited significantly higher proliferative and differentiation potential into adipocytes and chondrocytes, except for osteocytes. We propose the following conditions to improve MSC quality: (1) optimizing MNC seeding density to form single-cell colonies; (2) adjusting incubation times to increase highly proliferative MSCs; and (3) establishing a detachment processing time that excludes senescent cells.

Effectiveness of Soluble CTLA-4-Fc in the Inhibition of Bone Marrow T-Cell Activation in Context of Indoleamine 2.3-Dioxygenase (IDO) and CD4+Foxp3+ Treg Induction.

Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery. Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls. We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls. This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.

Day +60 WT1 assessment on CD34 selected bone marrow better predicts relapse and mortality after allogeneic stem cell transplantation in acute myeloid leukemia patients.

The aim of this study was to evaluate the role of WT1 expression after allogeneic stem cell transplantation (alloHSCT) in patients with acute myeloid leukemia (AML). We studied WT1 expression in bone marrow cells from 50 patients in complete remission on day +60 after transplant. WT1 was assessed on unfractionated bone marrow mononuclear cells (MNC) and on CD34+ selected cells (CD34+). A ROC curve analysis identified 800 WT1 copies on CD34+ selected cells, as the best cut-off predicting relapse (AUC 0.842, p=0.0006, 85.7% sensitivity and 81.6% specificity) and 100 copies in MNC (AUC 0.819, p=0.007, 83.3% sensitivity and 88.2% specificity). Using the 800 WT1 copy cut off in CD34+ cells, the 2 year cumulative incidence of relapse was 12% vs 38% (p=0.005), and 2 year survival 88% vs 55% (p=0.02). Using the 100 WT1 copy cut off in unfractionated MNC, the 2 year cumulative incidence of relapse 13% vs 44% (p=0.01) and the 2 year survival 88% vs 55% (p=0.08). In a multivariate Cox analysis WT1 expression in CD34 cells proved to highly predictive of relapse (p=0.004); also WT1 expression on unfractionated cells predicted relapse (p=0.03). In conclusion, day-60 WT1 expression after allogeneic HSCT is a significant predictor of relapse, particularly when tested on CD34+ selected bone marrow cells.

M2-like macrophages transplantation protects against the doxorubicin-induced heart failure via mitochondrial transfer

AimsThe alternatively activated macrophages have shown a cardioprotective effect in heart failure. However, the effect of M2 adoptive transfer in non-ischemic heart failure is unknown. In this study, we evaluated the efficacy of M-CSF plus IL-4 induced M2-like macrophages transplantation in doxorubicin-induced cardiotoxicity.MethodsBone marrow mononuclear cells were polarized as CCR2+CD206+ M2-like macrophages by a combination of M-CSF plus IL-4 treatment. C57BL/6 mice received a single intraperitoneal injection of doxorubicin (15 mg/kg). The treatment group were treated with M2-like macrophages (1 × 10^6 cells per mouse; i.v.) once a week for 2 weeks. After 3 weeks, we examined the percentage of resident cells and cardiac function. Furthermore, we evaluated cardiac fibrosis, cardiomyocyte apoptosis and circulating inflammatory factors. Finally, we investigated the mitochondria transfer in vitro in a direct and indirect co-culture conditions.ResultsCardiac function was significantly improved in doxorubicin-induced heart failure by adoptive transfer of M2-like macrophages. Besides, M2-like macrophages treatment attenuated cardiac fibrosis and cardiomyocyte apoptosis, as well as increased the level of circulating IL-4 and Th2 response. In vitro, M2-like macrophages could transfer mitochondria to injured cardiomyocytes in a direct and indirect way.ConclusionsIn our study, adoptive transfer of M2-like macrophages could protect against the doxorubicin-induced cardiotoxicity, which may be partly attributed to mitochondria transfer. And M2-like macrophages transplantation could become a treatment for non-ischemic heart failure in the clinical practice.Graphical

Improvement of bone marrow mononuclear cells cryopreservation methods to increase the efficiency of dendritic cell production

Cryopreservation is now considered an integral part of the biotechnological process, exploiting different types of cells and tissues in clinical practice. Among them, dendritic cells (DCs) deserve special attention, notably the immature tolerogenic cells (tolDCs), which provide natural tolerance in humans and animals. High cryolability of tolDCs has necessitated the search for the methods that would provide cryopreservation of their precursors; those more resistant to negative effects of cryopreservation factors, in particular, bone marrow or peripheral blood mononuclear cells (MNCs). Based on this, the aim of our research was to optimize the cryopreservation conditions for mice bone marrow MNCs with further assessment of their ability to form tolDCs ex vivo. A cryopreservation mode for bone marrow MNCs has been developed which provides structural and functional completeness of tolDCs obtained from them ex vivo. The ability of DCs derived from cryopreserved MNCs by the developed mode to induce T-regulatory (FOXP3+) cells in vitro when co-cultured with CD4+-lymphocytes was shown.Tolerogenic properties of the DCs derived from cryopreserved MNCs are implemented by increasing the content of hsp70 heat shock proteins and the expression rate of glucocorticoid-induced leucine zipper (GILZ). DCs with increased tolerogenic activities, obtained by the developed cryopreservation regimen, can be used in treatment of autoimmune diseases.In this research we not only evaluated the qualitative characteristics and tolerogenic activity of DCs produced in vitro from cryopreserved MNCs, but also outlined the prospects of accumulating their reserves in low-temperature banks for clinical use.

Tissue Engineering with Compact Bone-Derived Cell Spheroids Enables Bone Formation around Transplanted Tooth.

Although tooth transplantation is a desirable treatment option for congenital defects of permanent teeth in children, transplantation to a narrow alveolar ridge is not feasible. In this study, we investigated the possibility of bone tissue engineering simultaneously with tooth transplantation to enhance the width of the alveolar bone. Bone marrow mononuclear cells or cortical bone-derived mesenchymal stromal cell spheroids were seeded onto atelocollagen sponge and transplanted with freshly extracted molars from mice of the same strain. New bone formation around the tooth root was evaluated using micro-computed tomography and histological analysis. Tooth alone, or tooth with scaffold but without cells, was also transplanted and served as controls. Micro-computed tomography showed new bone formation in the furcation area in all four groups. Remarkable bone formation outside the root was also observed in the cortical bone-derived mesenchymal stromal cell group, but was scarce in the other three groups. Histological analysis revealed that the space between the new bone and the root was filled with collagen fibers in all four groups, indicating that the periodontal ligament was maintained. This study demonstrates the potential of simultaneous alveolar bone expansion employing bone tissue engineering approach using cortical bone-derived mesenchymal stromal cell spheroids for tooth transplantation. The use of an orthotopic transplantation model may further clarify the feasibility and functional recovery of the transplanted tooth over a longer period.

A Comparative Study of Different Stem Cell Transplantation for Spinal Cord Injury: A Systematic Review and Network Meta-Analysis

In the present study, we evaluated the efficacy and safety of different stem cell types for spinal cord injury (SCI) therapy to determine the superior treatment of SCI. A systematic literature search was performed using PubMed, Embase, the Cochrane Library, Web of Science, VIP, Chinese National Knowledge Infrastructure, and Wan Fang databases from initiation to January 30, 2021. A Bayesian network meta-analysis was performed using ADDIS (Aggregate Data Drug Information System) software. The PROSPERO registration number was CRD42020129635. We included 12 studies with 642 patients in the present study. A network meta-analysis revealed that bone mesenchymal stem cells (BMSCs) combined with rehabilitation training were significantly more effective than rehabilitation training alone in improving the American Spinal Injury Association (ASIA) impairment scale grade (odds ratio, 94.25; 95% confidence interval [CI], 6.71-9321.95), ASIA motor score (weighted mean difference [WMD], 6.67; 95% CI, 0.83-12.73), ASIA sensory functional score (WMD, 12.41; 95% CI, 3.42-21.72), and Barthel index (WMD, 7.24; 95% CI, 0.21-14.30). However, no statistically significant differences were observed between bone marrow mononuclear cells (MNCs) combined with rehabilitation training, umbilical cord-derived mesenchymal stem cells (UCMSCs) combined with rehabilitation training, or UCMSCs alone and rehabilitation alone for all indicators. In terms of safety, there were no serious and permanent adverse effects after transplantation of BMSCs, MNCs, or UCMSCs. BMSCs plus rehabilitation might be superior to other stem cell treatments of SCI in improving the ASIA impairment scale grade, ASIA motor score, ASIA sensory functional score, and Barthel index. The therapeutic effects of UCMSCs and MNCs remain to be confirmed.

Abstract LBA013: Phosphoproteomics reveals active drug targets on pathways of resistance and predicts response to midostaurin plus chemotherapy in FLT3 mutant-positive acute myeloid leukemia

Abstract Background: Midostaurin (mido) is approved for treatment of FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML). However, FLT3 mutation is not the only determinant of mido sensitivity. Here we report phosphoprotein signatures which predict response to chemotherapy (chemo) plus mido, and identify active drug targets on potential resistance pathways. Methods: Samples collected at diagnosis, post-treatment and relapse from FLT3+ patients treated with chemo+mido were obtained from the Leukemia Tissue Bank at the Princess Margaret Cancer Centre. Peptides and enriched phosphopeptides from bone marrow (BM) and peripheral blood (PB) mononuclear cells were quantified using liquid chromatography-tandem mass spectrometry. Signatures for BM/PB diagnosis samples were analyzed independently and used to train a classification machine learning algorithm to group patients (n=54) based on response to treatment. Additional features (e.g. genetic mutations) were also analyzed. Kaplan-Meier and Log-Rank test methods were used to assess differential survival between patient groups. To investigate pathways potentially driving resistance to chemo+mido, differential protein phosphorylation indexes were identified through comparison of post-treatment or relapse samples to paired diagnosis samples. To account for population heterogeneity, a filter was applied based on frequency of observation. Activated pathways potentially driving resistance were identified with functional enrichment tools and kinase-substrate enrichment analysis. Statistical significance of enrichment were determined using parametric methods and p-values adjusted for multiple testing using the Benjamini-Hochberg method. Results: Patients positive for a signature consisting of 26 phosphorylation sites showed a markedly longer survival time than negative patients (PB: 269 vs 76 weeks, Log-Rank p=1.30e-05; BM: 241 vs 56, Log-Rank p=2.13e-09). This signature partially overlapped with an ex-vivo signature of response to mido, described previously by Casado et al (Leukemia, 2018). A proteomic signature was also identified, with positive patients showing a longer survival time than negative patients (PB: 330 vs 173 weeks, Log-Rank p=5.0e-04; BM: 460 vs 156, Log-Rank p=5.2e-06). Key, diverging phosphorylation site signatures were identified between patients with refractory disease/early relapse and patients with complete response and no relapse or death within 2 years post-treatment. Pathways with increased activity in post-treatment or relapse specimens were associated with molecular functions such as regulation of cell proliferation, migration, differentiation and anti-apoptosis. Conclusions: We identified phosphoproteomic and proteomic signatures that differentiate survival mediated by response to chemo+mido. While the former was more predictive, both may enable further stratification of FLT3+ AML receiving mido treatment. Drug targets on pathways demonstrating increased activity in relapse/refractory cases may play a role in resistance; this will be determined in follow-up inhibitor studies. Citation Format: Luis Veiga Nobre, Celia Colomina Basanta, Salvatore Federico Pedicona, Arran David Dokal, Andrea Arruda, Ryan Smith, Calum Greenhalgh, Francesca Patella, Pedro Maria Casado-Izquierdo, Bela Wrench, Jane Theaker, Andrew Thompson, Mark D. Minden, John G. Gribben, David James Britton, Pedro Rodriguez Cutillas. Phosphoproteomics reveals active drug targets on pathways of resistance and predicts response to midostaurin plus chemotherapy in FLT3 mutant-positive acute myeloid leukemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA013.

In Vitro Model to Predict Response to Daratumumab Therapy in Relapsed/Refractory Multiple Myeloma

BackgroundSince its approval in 2015, daratumumab has become incorporated in standard-of-care regimens of antimyeloma therapy. However, response to daratumumab in patients with relapsed/refractory multiple myeloma (RRMM) is heterogeneous, and a reliable biomarker of response is lacking. Expression of CD38 was shown to be associated with response to daratumumab, but failed to predict responses consistently and is therefore not used in clinical practice.Aim of our study was to develop an in vitro method that accurately identifies patients with RRMM that will respond to daratumumab treatment.MethodsWe obtained CD38 mean fluorescent intensity (MFI) of plasma cells from patients with RRMM just before starting daratumumab therapy, as well as best clinical response to treatment and duration of response to daratumumab in weeks. When possible, extra material was obtained at the bone marrow biopsy performed before the start of daratumumab treatment and frozen in liquid nitrogen in our local biobank facility. All included patients provided written informed consent.Bone marrow mononuclear cells from patients with RRMM were thawed and cultured for a week in a hydrogel-based culture system, supplemented with pro-survival cytokines IL-6 and APRIL. To examine the in vitro response to daratumumab, we added 0.1 μg/ml daratumumab or 0.1 μg/ml IgG1κ isotype to the culture wells. We measured complement-dependent cytotoxicity (CDC) by adding 10% pooled human serum and antibody-dependent cellular cytotoxicity (ADCC) by adding healthy-donor peripheral blood mononuclear cells (PBMC) in a 10:1 effector-to-target ratio. After overnight incubation, hydrogel cultures were broken down mechanically and specific lysis was determined with flow cytometry, using CD138 and CD38 multi-epitope antibodies to select plasma cells, and using ToPro3 live/dead stain and Flow-Count Fluorospheres to obtain absolute numbers of surviving plasma cells.ResultsFirst, we analyzed CD38 MFI of plasma cells from 35 patients with RRMM just before starting daratumumab treatment, both as monotherapy or as part of a regimen. As was previously reported by others 1,2, we found a significant association between expression of CD38 and response (p = 0.01) (Figure 1A). However, we also observed considerable overlap between CD38 MFI values of responders and non-responders, with AUC ROC = 0.75. Therefore the discriminatory value of CD38 expression to predict response is weak.Next, we tested daratumumab in vitro on plasma cells obtained from 10 patients with RRMM. Daratumumab ADCC was associated with clinical response by the corresponding patients (p = 0.008), with a median specific lysis of 54.3% for patients who obtained a PR and 26.0% for patients who did not obtain a PR (Figure 1B). Median CDC for patients who did not obtain a PR was 3.16% versus 45.3% for patients who did obtain a PR, without a significant difference between the two groups (p = 0.18). Further evaluation of these results revealed AUC ROC of 1.00 and 0.81 for ADCC and CDC, respectively, outperforming CD38 expression. For ADCC, a threshold value of 39.6% lysis translated to a diagnostic accuracy of 100%, while for CDC a threshold value of 29.4% resulted in a diagnostic accuracy of 87.5%. Ultimately, we compared these results to CD38 expression. With an optimal CD38 MFI threshold value of 10989, diagnostic accuracy of CD38 expression was 71.4%. In addition, we examined ability of the test methods to predict the clinically experienced duration of response to daratumumab treatment. We observed no difference in duration of response when using the optimal threshold values of CD38 MFI (p = 0.13) and CDC lysis (p = 0.12), but duration of response was significantly different for patients with an ADCC lysis above and below the threshold value of 39.6% (p = 0.002) (Figure 1C).ConclusionIn summary, we developed an straightforward and consistent in vitro method to predict response to daratumumab treatment in vivo. Measuring ADCC in patient-derived plasma cells using healthy-donor PBMC within our hydrogel-based culture system corresponds with the overall clinical response and duration of response to daratumumab. Our results support further clinical validation of the in vitro use of primary plasma cells to identify patients most likely to benefit from treatment.

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Evolving Exhaustion of T Cells during the Course of the Disease in AML Can be Abrogated By CD33 BiTE ® Construct Mediated Cytotoxicity

Characterisation of the Tumour Proteome in Primary Extramedullary Multiple Myeloma Identifies Key Proteins Associated with Transendothelial Migration

Introduction: Extramedullary multiple myeloma (EMM) refers to the spread of clonal plasma cells to tissues extending outside of the bone marrow microenvironment. EMM is present at the time of diagnosis in 6-10% of patients, however, this increases to 13-26% in patients with disease progression and relapse. Cancer cells are suspected to spread to new tissues and organs via the circulatory system as a result of molecular changes that allow malignant cells to escape the bone marrow (BM). For example, downregulation of CXCR4 (C-X-C Motif Chemokine Receptor 4), an important factor in cellular homing to the BM, has frequently been reported to be linked to the EMM phenotype. In addition, the majority of patients presenting with EMM have highly complex cytogenetic abnormalities and high-risk cytogenetic markers such as t(14;16). As EMM is an indicator of a more aggressive disease, more intensive treatment, including combination chemotherapy, is often recommended. Many of the underlying molecular mechanisms accompanying EMM are yet to be characterised. Our mass-spectrometry (MS)-based proteomic study provides insight into the unique molecular mechanisms associated with EMM, identifying key proteins linked to the progression of medullary multiple myeloma (MM) to EMM.Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender matched medullary MM (n=8) and EMM (n=9) bone-marrow derived mononuclear cells (MNCs) was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. For protein identification, the following search parameters were used: (i) 0.02 Da for MS/MS mass tolerance, (ii) 10 ppm for peptide mass tolerance, (iii) variable modification settings for methionine oxidation, (iv) fixed modification settings in relation to carbamido-methylation and (v) tolerance for up to two missed cleavages. Peptide probability was set to high confidence. Datasets were imported into Progenesis QI (version 2.0) software for further analysis. Data was filtered based on an ANOVA p-value of ≤0.05, fold change >1.5 between experimental groups, and proteins with ≥1 unique peptides contributing to the identification. Proteins with less than 70% valid values were removed from the analysis. G:profiler and STRING were utilized for functional enrichment and the characterisation of protein interaction patterns.Results: Our quantitative MS-based proteomic analysis identified a total of 492 proteins with significantly altered abundances between EMM and MM bone marrow MNC. Of these significant proteins, 275 were found to be increased in EMM compared to medullary MM and 217 were found to be decreased in EMM compared to medullary MM. Hierarchical clustering was performed to highlight the proteomic profile associated with extramedullary disease (Figure 1A). KEGG pathway analysis and gene ontology (GO) analysis of proteins found to be increased in EMM indicated an increase in proteins associated with cell adhesion, invasion, and migration pathways (Figure 1B). Interestingly, several proteins involved in leukocyte transendothelial migration were significantly increased in EMM indicating their potential involvement in the dissemination of MM cells from the bone marrow microenvironment to distal tissues (Figure 1C). Among the proteins found to be involved in this biological pathway was junctional adhesion molecule-A (F11R), a protein previously reported to play a role in EMD pathophysiology [1]. Other proteins involved in MM invasion and migration including Rho-associated protein kinase 2 (ROCK2), Ras-related C3 botulinum toxin substrate 1 (Rac1) and platelet endothelial cell adhesion molecule (PECAM-1) were significantly increased in EMM.Conclusion: Using high-resolution mass spectrometry to characterise the tumour proteome of MM patients with extramedullary disease, we have identified a significant increase in the abundance of proteins associated with leukocyte transendothelial invasion in primary EMM samples. Our study provides further insight into the molecular mechanisms within EMM and thus holds potential to enhance current efforts to provide a more personalised therapeutic approach for EMM patients.

Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stem Cells from Sheep: Culture Characteristics.

Simple SummaryMesenchymal stem cells (MSCs) are available in minuscule numbers in the body or placental tissues. These cells have mostly been harvested from bone marrow and adipose tissue. To broaden the currently available knowledge, the current study provides (a) information on the feasibility of isolation of MSCs at different ambient temperatures, (b) details of MSCs’ culture characteristics with respect to the physiological status of the donor, and (c) information on the viability of cryopreserved cells. Bone marrow harbors a higher mononuclear cell fraction than that of the adipose tissue, although percent adherent cells are comparably more in adipose tissue. MSCs from a pregnant donor show enhanced proliferation and differentiation potential, although further studies are desired. The cryopreserved cells have comparable characteristics to that of the fresh cells. In conclusion, donor animals’ tissue type and physiological status may affect MSCs’ characteristics and should be taken into consideration while applying in clinical settings.The current study demonstrates the culture characteristics of adipose tissue and bone marrow-derived mesenchymal stem cells (MSC). The study evaluates the effect of ambient temperature, physiological status of the donor and the tissue source on sheep (Ovis aries) mesenchymal stem cells. The tissue samples were harvested from full term pregnant female sheep (n = 9) and male sheep (n = 10). Adipose tissue was harvested from n = 9 sheep and bone marrow from n = 10 sheep. The samples (adipose tissue, n = 2; bone marrow, n = 3) transported at cold ambient temperature (<10 °C) failed to yield MSCs while those (n = 14) at higher (>20 °C) ambient temperature successfully yielded MSCs. Bone marrow mononuclear cell (MNC) fraction was higher than the adipose tissue-derived stromal vascular fraction (SVF), but the percent adherent cells (PAC) was higher in the later cell fraction. Adipose tissue-derived MSCs from the full term female sheep had a significantly (p < 0.05) higher proliferation potential as compared to those of the male sheep-derived MSCs. Female sheep MSCs also had rapid differentiation potential. The cryopreserved MSCs had morphological features comparable to that of the fresh cells. In conclusion, the tissue type and physiological status of donor animal may affect MSCs’ characteristics and should be taken into consideration while applying in clinical settings.

Rituximab and High-Dose Methylprednisolone for the Initial Treatment of Chronic Lymphocytic Leukemia Is Associated with Promising Clinical Activity and Minimal Hematologic Toxicity

Myelosuppression is associated with significant morbidity and mortality in chronic lymphocytic leukemia (CLL) and is often exacerbated by currently available treatments for this disease. Myelosuppression often requires dose reductions in cytotoxic agents, delays in therapy, truncated treatment courses, or the exclusion of elderly or infirmed individuals from receiving standard chemoimmunotherapy.High-dose methylprednisolone (HDMP) and the anti-CD20 mAb rituximab (Rituxan), each is non-myelotoxic and can induce partial remissions when used alone to treat patients with CLL. However, complete remissions (CRs) in CLL patients are rarely observed following treatment with either agent. Recently, we reported the use of the combination of HDMP and rituximab in fludarabine-refractory patients [Castro Leukemia 2008]. The promising clinical activity and tolerability of this regimen in the refractory setting led to the evaluation of this regimen for the initial treatment of patients with CLL. To mitigate toxicity associated with use of HDMP, we employed a 40% reduction in the dose of HDMP by administering solumedrol at 1 gm/m2 for 3 instead of 5 days during each 4-week treatment cycle. Rituximab was given weekly for 12 weeks at 375mg/m2 or at 750mg/m2 in 9 doses over the 12 weeks of treatment to chemotherapy na·ve CLL patients with an NCI-WG indication for treatment. Objectives were to determine the response rate, safety, and toxicity of this regimen. PFS, overall survival (OS), hematologic, and immune parameters were also evaluated. We enrolled 28 patients who had a median age 65 years (range 48–80), 29% of whom were at least 70 years old. Forty-six were high risk by modified Rai classification, 39% had unfavorable cytogenetic features, and 64% had CLL cells that used unmutated IgVH region genes or exhibited high expression levels of ZAP-70. Patients received the full course of treatment without dose reductions or delays and the treatment well tolerated. Overall response rate was 96% with complete remission achieved in 32% of patients, 22% of whom had no detectable minimal residual disease (MRD) by sensitive 4-color flow cytometry capable of detecting residual CLL cells when present in marrow mononuclear cells at levels of more than 0.01%. Those patients over age 70 had an ORR of 100% with 38% CR, and 11% MRD-negative CR. There was a non-significant trend towards those receiving rituximab at 750mg/m2 being more likely to achieve a CR to treatment (Odds Ratio (OR) = 4.3, p-value 0.09). Individually, both beta-2-microglobulin (p=0.04) and splenomegaly palpable to at least 5 cm below the left costal margin (p<0.01) were associated with failure to achieve a CR to HDMP and rituximab. However, multivariable logistic regression analysis identified only splenomegaly as being significantly associated with failure to achieve a CR (OR = 0.096, p value = 0.03) to this regimen.The majority of adverse events were grade I/ II (80%) and hematologic toxicity was minimal. During the course of treatment, we noted resolution of thrombocytopenia or anemia. (See Figure) A total of 81 cycles of treatment were administered and only one red cell transfusion and no platelet transfusions were required. Serum immunoglobulins decreased with therapy and a low threshold was used to initiate intravenous gammaglobulin for those patients with experiencing recurrent infections. Percent of CD4 T cell counts increased significantly from median 2% (+/−4% 95% CI) to 32% (+/−8%) (Student t-test p < 0.0001). All patients had a CD4 count of greater than 200 cells/μl (median 848 cells/ul) two months after the completion of treatment.Six patients received consolidation with alemtuzumab at least 4 months following initial therapy and 83% of these patients became MRD negative. The CR and MRD negative CR rate following HDMP and Rituximab with 6 patients receiving consolidation were 43% and 25% respectively. With over three years of follow-up (36.3 mo) median PFS and time to requiring additional therapy have not been reached and OS is 96%. Achieving a MRD negative response was associated with significantly prolonged PFS and time to requiring additional therapy over all other responses (log-rank test p= 0.036 and 0.016 respectively).In summary, the combination of HDMP and rituximab is an effective non-myelotoxic treatment regimen for patients with CLL. Treatment was well tolerated, notably in elderly patients and those with baseline cytopenias making this regimen an effective and safe alternative for frontline therapy. [Display omitted]

In Utero Injections of Nanoparticle Encapsulated Myelin Oligodendrocyte Glycoprotein Peptides Prevent Experimental Autoimmune Encephalopathy Via Central Tolerance

IntroductionIn utero hematopoietic cell transplantation (IUHCT) of allogenic bone marrow mononuclear cells (MNC) results in successful engraftment across immune barriers. This is secondary to immune tolerance that is acquired due to the presence of donor cells in the thymus prior to E17. We have demonstrated that immune tolerance to specific proteins is possible by injecting nanoparticle encapsulated peptides which are taken up by dendritic cells and presented in the thymus.The aim of this study is to show that in utero injections of nanoparticle encapsulated antigen can produce tolerance to that antigen. We demonstrate this by preventing the autoimmune disease Experimental Autoimmune Encephalopathy (EAE, the mouse equivalent of multiple sclerosis) by injecting NPs loaded with the peptide Myelin Oligodendrocyte Glycoprotein (35-55) (MOG35-55.)MethodsIn utero injections of nanoparticles were performed in C57BL/6 mice at E14 intravenously through the vitelline vein. Post-natal boosters were injected subcutaneously at 2, 4, 6, and 8 weeks of age. Disease inducing injections of MOG with Complete Freud's Adjuvant (CFA) was given at 8 1/7 weeks. Intraperitoneal pertussis toxin (PT) was given on the same day as the MOG/CFA and again 48 hours later. Positive controls got MOG/CFA and PT injections. Negative controls got CFA without MOG as well as the PT injections.Mice were monitored for paralysis using the 5-point EAE scoring system for 4 weeks after the MOG/CFA injection. Spines and brains were harvested 2 weeks after the disease inducing MOG/CFA injection. Spines and brains were filtered and then MNCs were isolated using a Percoll gradient. MNCs were stained for CD45, CD19, CD11b, CD3, CD4, CD8, and I-A(b) MOG35-55 tetramer. Cells were then analyzed on the FACSAria™.Results are expressed as mean±SEM, and statistical analysis was performed using 1-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children's Hospital of Philadelphia.ResultsMice that received MOG nanoparticles prenatally along with boosters (Group A) developed disease with less frequency than mice that received MOG nanoparticles postnatally along with boosters (Group B) or positive controls (Group D) (52.0% vs 87.5% vs 90.0% p<0.005) supporting the theory that antigen needs to be expressed prior to E17 in the thymus to develop tolerance. Mice that were injected prenatally with MOG nanoparticles and did not receive booster injections (Group C) also did as poorly as positive controls (85.72% vs 90.0%) suggesting that continued antigen expression is necessary to maintain tolerance. Group A also had significantly lower EAE scores on days 15 to 28 compared to Group B, Group C, and the positive control group (p<0.005).Spinal cord and brain analysis of the Group A mice showed significantly fewer CD45 cells compared to the positive control but not the negative control (133.2±52.0 vs 6062.5±1166.9 vs 79.8±15.0 p<0.0001.) The same is seen with CD3 cells (45.6±17.0 vs 4727.3±1283.0 vs 21.3±2.8 p<0.0001) and CD4 cells (12.6±5.7 vs 3339.6912.1 vs 11.8±0.8 p<0.0001.) Finally, the same is seen with CD4+ Tetramer+ cells (0.7±0.3 vs 379.1±156.6 vs 2.9±1.0 p<0.0001) showing that there is central tolerance to the MOG peptide.ConclusionBy decreasing the frequency and average severity of EAE and demonstrating the lack of CD4+ Tetramer+ cells in the spine, we have shown that autoimmune disease can be prevented by central tolerance to the target antigen. We have demonstrated that this central tolerance is possible with prenatal injections of nanoparticle encapsulated peptide. We have also shown that continued antigen presentation is necessary to maintain central tolerance. We hope that nanoparticle encapsulated peptides could provide a possible pharmacological benefit for numerous autoimmune diseases in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Daratumumab Activity Is Agnostic to Fc Gamma Receptor 3A (FcγR3A) 158 Genotype in Patients with Primary Refractory or Relapsed Systemic AL Amyloidosis

Introduction: Treatment targeted at the clonal plasma cells that produce the pathologic immunoglobulin light chain in patients with systemic AL amyloidosis, if effective and timely, can normalize serum free light chains resulting in organ responses and significantly improved overall survival. A third of patients with AL have no response to initial therapy, and over half of those who respond to initial therapy will relapse in 4 years; non-responders and relapsed patients with cardiac involvement are at especially high risk of shortened survival. Single agent daratumumab has been shown to have a response rate of 36% in relapsed refractory multiple myeloma patients (Lokhorst, Plesner et al. 2015). Daratumumab is an option for patients with AL who have marrow plasma cell burden > 10%, consistent with myeloma. Antibody- dependent cellular cytotoxicity is a mechanism of action of daratumumab, and this implicates recognition of the FC portion of the therapeutic antibody by FC receptors on effector cells such as macrophages and NK cells. A single nucleotide polymorphism at codon 158 of the FcγR3A receptor has been shown to affect the therapeutic efficacy of monoclonal antibodies(Mellor, Brown et al. 2013). The aim of this study was to assess hematologic and organ responses to daratumumab in a cohort of AL patients with primary refractory or relapsed clonal plasma cell disease and to test whether FcγR3A genotype influenced response to daratumumab.Materials and Methods: Patients with biopsy proven AL amyloidosis and organ involvement evaluated at Tufts Medical Center who received daratumumab were identified and evaluated for hematologic and organ responses according to consensus criteria (Comenzo, Reece et al. 2012, Palladini, Hegenbart et al. 2014). FcγR3a genotype for each patient was identified by PCR amplification and sequencing using genomic DNA from blood or marrow mononuclear cells. The translation of 158F or 158V in the alleles of the membrane-proximal extracellular domain was determined by T/G zygosity at nt559 in the gene sequence.Results: Fifteen patients with AL amyloidosis (λ=10, κ=5) who had primary refractory (no response after at least 2 cycles of initial therapy) (n = 5) or relapsed clonal plasma cell disease (n = 10) received daratumumab at standard doses at a median of 29 months from diagnosis (range, 2-107). Median age at diagnosis was 62 years (47-73) and 60% were male. The median lines of prior therapy were 3 (1-5). Eighty seven percent (n=13) had received bortezomib-based therapy and 53% (n=8) had undergone high-dose melphalan with autologous stem cell transplant. Nine patients had renal involvement (6 with stage 3), and 9 had cardiac involvement with a median NT-proBNP of 2373pg/ml (553-18021). All patients are alive with a median follow-up of 7 months (1-16). Hematologic responses were seen in 87% (CR=5, VPGR=8) with a median time to best response of 71 days. Forty four percent (n=4) had a renal response within an average of 107 days while 55% (n=5) achieved a cardiac response within an average of 149 days. Responses did not correlate with FcγR3A genotype (see figure below). Those with 158V/V or V/F had 3 CR, 3 VGPR and 1 no response (NR) while those with 158F/F had 2 CR, 5 VGPR and 1 NR. The ratio of FcγR3A 158 genotypes in patients with systemic AL amyloidosis has not previously been studied.Conclusions: Daratumumab achieved rapid hematologic responses in over 85% of patients with primary refractory or relapsed systemic AL amyloidosis with significant organ responses. Daratumumab activity was agnostic to FcγR3A 158 genotypes in this cohort of patients, in whom, of note, the distribution of genotypes is of interest. The basis for daratumumab activity may be linked to other mechanisms such as direct cytotoxicity or to the low fraction of cells in S-phase in AL plasma cell clones (Gertz, Kyle et al. 1989). Daratumumab is a very active agent in AL and its rapid clinical evaluation in patients with newly diagnosed AL is anticipated. [Display omitted] DisclosuresComenzo:Prothena: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Karyopharm: Research Funding; Takeda: Research Funding.

HOTAIR expression and prognostic impact in acute myeloid leukemia patients

BackgroundAcute myeloid leukemia (AML) is a disorder characterized by a rapid onset of symptoms attributable to bone marrow failure due to clonal proliferation of primitive hematopoietic stem cells or progenitor cells. Epigenetic abnormalities play an important role in the development and progression of acute leukemia. Long non-coding ribonucleic acid (lncRNA) plays an important role in epigenetic regulation. Homeobox (Hox) transcript antisense intergenic RNA (HOTAIR) is a lncRNA which has been determined to be a negative prognostic indicator in various solid-tumor patients. However, its role in hematopoietic tumors as AML is to be assessed. This study aimed at measuring lncRNA HOTAIR expression level on bone marrow (BM) mononuclear cells in newly diagnosed AML patients and correlating its expression with their outcome and different prognostic variables. This provides new prospective for a novel marker involved in development and progression of AML which can be used as a diagnostic marker and a target of therapy. The current study included 65 subjects divided into 35 newly diagnosed AML adult patients (before initiation of chemotherapy) and 30 non-leukemic adult patients who are candidates for BM aspiration for causes other than hematological malignancies as immune thrombocytopenic purpura and hypersplenism as controls. HOTAIR expression was measured on BM mononuclear cells by quantitative reverse transcription polymerase chain reaction (qRT-PCR).ResultsHOTAIR expression was found to be significantly upregulated in AML patients (probability (p) value = 0.000) and it can be used as a diagnostic biomarker of AML as confirmed by a significant difference between cases and controls using receiver operating characteristic curve (ROC) analysis. However, it was not significantly correlated with event free survival (EFS) or prognostic variables.ConclusionThis study showed that the expression of HOTAIR is upregulated in de novo AML patients and can be used as a diagnostic marker. However, highly expressed HOTAIR is not associated with poor prognosis.