Delineating functional and molecular impact of ex vivo sample handling in precision medicine.

Consistent handling of samples is crucial for achieving reproducible molecular and functional testing results in translational research. Here, we used 229 acute myeloid leukemia (AML) patient samples to assess the impact of sample handling on high-throughput functional drug testing, mass spectrometry-based proteomics, and flow cytometry. Our data revealed novel and previously described changes in cell phenotype and drug response dependent on sample biobanking. Specifically, myeloid cells with a CD117 (c-KIT) positive phenotype decreased after biobanking, potentially distorting cell population representations and affecting drugs targeting these cells. Additionally, highly granular AML cell numbers decreased after freezing. Secondly, protein expression levels, as well as sensitivity to drugs targeting cell proliferation, metabolism, tyrosine kinases (e.g., JAK, KIT, FLT3), and BH3 mimetics were notably affected by biobanking. Moreover, drug response profiles of paired fresh and frozen samples showed that freezing samples can lead to systematic errors in drug sensitivity scores. While a high correlation between fresh and frozen for the entire drug library was observed, freezing cells had a considerable impact at an individual level, which could influence outcomes in translational studies. Our study highlights conditions where standardization is needed to improve reproducibility, and where validation of data generated from biobanked cohorts may be particularly important.

Targeting autophagy as a therapeutic strategy in pediatric acute lymphoblastic leukemia.

Autophagy is activated in response to a variety of stress conditions including anti-cancer therapies, and tumors cells often depend on autophagy for survival. In this study, we have evaluated inhibition of autophagy as therapeutic strategy in acute lymphoblastic leukemia (ALL) in children, both as a single treatment and in combination with glucocorticoid (GC) Dexamethasone (Dexa). Analysis of proteomics and RNA-seq of ALL cell lines and primary samples identified an upregulation of Vps34 and ATG14 proteins and autophagy and lysosomal pathway enrichment in a genetic subgroup with a recurrent t(12;21) translocation. Cells from this sugbroup were also significantly more sensitive to the selective autophagy or lysosomal inhibitors than cells with other genetic rearrangements. Further, combination of Dexa with either lysosomal or autophagy inhibitors was either synergistic or additive in killing leukemic cells across various genetic and lineage backgrounds, for both cell lines and primary samples, as assessed using viability assays and SynergyFinder as well as apoptotic caspase 3/7-based live-cell assays. Our data demonstrate that targeting autophagy represents a promising strategy for the treatment of pediatric ALL, both as a selective modality for the t(12;21) pre-B-ALL subgroup, and in combination treatments to sensitize to GC-induced cytotoxicity.

Novel precision medicine approaches and treatment strategies in hematological malignancies.

Genetic testing has been applied for decades in clinical routine diagnostics of hematological malignancies to improve disease (sub)classification, prognostication, patient management, and survival. In recent classifications of hematological malignancies, disease subtypes are defined by key recurrent genetic alterations detected by conventional methods (i.e., cytogenetics, fluorescence in situ hybridization, and targeted sequencing). Hematological malignancies were also one of the first disease areas in which targeted therapies were introduced, the prime example being BCR::ABL1 inhibitors, followed by an increasing number of targeted inhibitors hitting the Achilles' heel of each disease, resulting in a clear patient benefit. Owing to the technical advances in high-throughput sequencing, we can now apply broad genomic tests, including comprehensive gene panels or whole-genome and whole-transcriptome sequencing, to identify clinically important diagnostic, prognostic, and predictive markers. In this review, we give examples of how precision diagnostics has been implemented to guide treatment selection and improve survival in myeloid (myelodysplastic syndromes and acute myeloid leukemia) and lymphoid malignancies (acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and chronic lymphocytic leukemia). We discuss the relevance and potential of monitoring measurable residual disease using ultra-sensitive techniques to assess therapy response and detect early relapses. Finally, we bring up the promising avenue of functional precision medicine, combining ex vivo drug screening with various omics technologies, to provide novel treatment options for patients with advanced disease. Although we are only in the beginning of the field of precision hematology, we foresee rapid development with new types of diagnostics and treatment strategies becoming available to the benefit of our patients.

Prediction model for drug response of acute myeloid leukemia patients.

Despite some encouraging successes, predicting the therapy response of acute myeloid leukemia (AML) patients remains highly challenging due to tumor heterogeneity. Here we aim to develop and validate MDREAM, a robust ensemble-based prediction model for drug response in AML based on an integration of omics data, including mutations and gene expression, and large-scale drug testing. Briefly, MDREAM is first trained in the BeatAML cohort (n = 278), and then validated in the BeatAML (n = 183) and two external cohorts, including a Swedish AML cohort (n = 45) and a relapsed/refractory acute leukemia cohort (n = 12). The final prediction is based on 122 ensemble models, each corresponding to a drug. A confidence score metric is used to convey the uncertainty of predictions; among predictions with a confidence score >0.75, the validated proportion of good responders is 77%. The Spearman correlations between the predicted and the observed drug response are 0.68 (95% CI: [0.64, 0.68]) in the BeatAML validation set, -0.49 (95% CI: [-0.53, -0.44]) in the Swedish cohort and 0.59 (95% CI: [0.51, 0.67]) in the relapsed/refractory cohort. A web-based implementation of MDREAM is publicly available at https://www.meb.ki.se/shiny/truvu/MDREAM/ .

Immunoregulatory effects of RGMb in gut inflammation.

Graft-versus-host disease (GvHD) is a major complication after allogeneic hematopoietic cell transplantation (HCT). Current strategies to prevent GvHD with immunosuppressive drugs carry significant morbidity and may affect the graft-versus-tumor (GVT) effect. Inflammatory bowel disease (IBD) is an intestinal inflammatory condition that affects more than 2 million people in the United States. Current strategies to prevent colitis with immunosuppressive drugs carry significant morbidity. Recently, Repulsive Guidance Molecule b (RGMb) has been identified as part of a signaling hub with neogenin and BMP receptors in mice and humans. In addition, RGMb binds BMP-2/4 in mice and humans as well as PD-L2 in mice. RGMb is expressed in the gut epithelium and by antigen presenting cells, and we found significantly increased expression in mouse small intestine after total body irradiation HCT conditioning. We hypothesized that RGMb may play a role in GvHD and IBD pathogenesis by contributing to mucosal inflammation. Using major-mismatched HCT mouse models, treatment with an anti-RGMb monoclonal antibody (mAb) that blocks the interaction with BMP-2/4 and neogenin prevented GvHD and improved survival compared to isotype control (75% versus 30% survival at 60 days after transplantation). The GVT effect was retained in tumor models. Using an inflammatory bowel disease dextran sulfate sodium model, treatment with anti-RGMb blocking monoclonal antibody but not isotype control prevented colitis and improved survival compared to control (73% versus 33% at 21 days after treatment) restoring gut homeostasis. Anti-RGMb mAb (9D1) treatment decreased IFN-γ and significantly increased IL-5 and IL-10 in the gut of the treated mice compared to the isotype control treated mice.

Targeting SAMHD1 with hydroxyurea in first-line cytarabine-based therapy of newly diagnosed acute myeloid leukaemia: Results from the HEAT-AML trial.

BACKGROUND: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.

Integrative multi-omics and drug response profiling of childhood acute lymphoblastic leukemia cell lines.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/forall .

MTH1 Inhibitor TH1579 Induces Oxidative DNA Damage and Mitotic Arrest in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy, exhibiting high levels of reactive oxygen species (ROS). ROS levels have been suggested to drive leukemogenesis and is thus a potential novel target for treating AML. MTH1 prevents incorporation of oxidized nucleotides into the DNA to maintain genome integrity and is upregulated in many cancers. Here we demonstrate that hematologic cancers are highly sensitive to MTH1 inhibitor TH1579 (karonudib). A functional precision medicine ex vivo screen in primary AML bone marrow samples demonstrated a broad response profile of TH1579, independent of the genomic alteration of AML, resembling the response profile of the standard-of-care treatments cytarabine and doxorubicin. Furthermore, TH1579 killed primary human AML blast cells (CD45+) as well as chemotherapy resistance leukemic stem cells (CD45+Lin-CD34+CD38-), which are often responsible for AML progression. TH1579 killed AML cells by causing mitotic arrest, elevating intracellular ROS levels, and enhancing oxidative DNA damage. TH1579 showed a significant therapeutic window, was well tolerated in animals, and could be combined with standard-of-care treatments to further improve efficacy. TH1579 significantly improved survival in two different AML disease models in vivo. In conclusion, the preclinical data presented here support that TH1579 is a promising novel anticancer agent for AML, providing a rationale to investigate the clinical usefulness of TH1579 in AML in an ongoing clinical phase I trial. SIGNIFICANCE: The MTH1 inhibitor TH1579 is a potential novel AML treatment, targeting both blasts and the pivotal leukemic stem cells while sparing normal bone marrow cells.

The transcriptome-wide landscape of molecular subtype-specific mRNA expression profiles in acute myeloid leukemia.

Molecular classification of acute myeloid leukemia (AML) aids prognostic stratification and clinical management. Our aim in this study is to identify transcriptome-wide mRNAs that are specific to each of the molecular subtypes of AML. We analyzed RNA-sequencing data of 955 AML samples from three cohorts, including the BeatAML project, the Cancer Genome Atlas, and a cohort of Swedish patients to provide a comprehensive transcriptome-wide view of subtype-specific mRNA expression. We identified 729 subtype-specific mRNAs, discovered in the BeatAML project and validated in the other two cohorts. Using unique proteomics data, we also validated the presence of subtype-specific mRNAs at the protein level, yielding a rich collection of potential protein-based biomarkers for the AML community. To enable the exploration of subtype-specific mRNA expression by the broader scientific community, we provide an interactive resource to the public.

High-parametric evaluation of human invariant natural killer T cells to delineate heterogeneity in allo- and autoimmunity.

Human invariant natural killer T (iNKT) cells are a rare innate-like lymphocyte population that recognizes glycolipids presented on CD1d. Studies in mice have shown that these cells are heterogeneous and are capable of enacting diverse functions, and the composition of iNKT cell subsets can alter disease outcomes. In contrast, far less is known about how heterogeneity in human iNKT cells relates to disease. To address this, we used a high-dimensional, data-driven approach to devise a framework for parsing human iNKT heterogeneity. Our data revealed novel and previously described iNKT cell phenotypes with distinct functions. In particular, we found 2 phenotypes of interest: (1) a population with T helper 1 function that was increased with iNKT activation characterized by HLA-II+CD161- expression, and (2) a population with enhanced cytotoxic function characterized by CD4-CD94+ expression. These populations correlate with acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation and with new onset type 1 diabetes, respectively. Our study identifies human iNKT cell phenotypes associated with human disease that could aid in the development of biomarkers or therapeutics targeting iNKT cells.

Blockade of TIM-1 on the donor graft ameliorates graft-versus-host disease following hematopoietic cell transplantation.

Acute graft-versus-host disease (GVHD) is a leading cause of mortality after allogeneic hematopoietic cell transplantation (HCT) mediated by dysregulated T-cell immune reconstitution. Given the role of the T-cell immunoglobulin and mucin 1 (TIM-1) surface protein in many immune processes, including organ transplantation tolerance, we asked if TIM-1 might drive post-transplant inflammation and acute GVHD. TIM-1 binds to phosphatidylserine (PtdSer), and agonism of TIM1 on immune cells is proinflammatory. HCT conditioning results in a significant supply of PtdSer from apoptosis and cellular debris. Using murine models, treatment with an antagonistic anti-TIM-1 monoclonal antibody (mAb) protects against acute GVHD while maintaining graft-versus-tumor effects. In contrast, the addition of exogenous free PtdSer worsened GVHD in a TIM-1-dependent manner. Importantly, TIM-1 blockade did not alter the expansion of donor T cells in vitro or in vivo. Instead, TIM-1 blockade reduces proinflammatory cytokines and promotes anti-inflammatory factors like carbonic anhydrase 1 and serum amyloid A1 in the gut tissue. This is mediated by TIM-1 on donor cells, as HCT of wild-type (WT) bone marrow (BM) and conventional T (Tcon) cells into TIM-1-/- knockout (KO) recipient mice showed little survival advantage compared with WT recipients, whereas WT recipients of TIM-1-/- KO Tcon cells or TIM1-/- KO BM had improved survival, in part due to the expression of TIM-1 on donor invariant natural killer T cells, which drives inflammation. Finally, in a humanized mouse xenograft GVHD model, treatment with anti-human TIM-1 antagonist mAb reduced GVHD disease burden and mortality. This supports TIM-1 as important for GVHD pathogenesis and as a target for the prevention of GVHD.

T cells expressing chimeric antigen receptor promote immune tolerance.

Cellular therapies based on permanent genetic modification of conventional T cells have emerged as a promising strategy for cancer. However, it remains unknown if modification of T cell subsets, such as Tregs, could be useful in other settings, such as allograft transplantation. Here, we use a modular system based on a chimeric antigen receptor (CAR) that binds covalently modified mAbs to control Treg activation in vivo. Transient expression of this mAb-directed CAR (mAbCAR) in Tregs permitted Treg targeting to specific tissue sites and mitigated allograft responses, such as graft-versus-host disease. mAbCAR Tregs targeted to MHC class I proteins on allografts prolonged islet allograft survival and also prolonged the survival of secondary skin grafts specifically matched to the original islet allograft. Thus, transient genetic modification to produce mAbCAR T cells led to durable immune modulation, suggesting therapeutic targeting strategies for controlling alloreactivity in settings such as organ or tissue transplantation.

MAIT cells accumulate in placental intervillous space and display a highly cytotoxic phenotype upon bacterial stimulation.

During pregnancy, the maternal immune system must tolerate the developing foetus, and yet retain a potent antimicrobial response to prevent infections. Mucosal associated invariant T (MAIT) cells recognize microbial-derived vitamin B metabolites presented on the MR1 molecule, but their presence and function at the foetal-maternal interface is not known. We here isolated mononuclear cells from paired samples of peripheral blood (PB), intervillous blood (IVB), and decidua parietalis (DP) following uncomplicated term pregnancies. Interestingly, MAIT cells were highly enriched in IVB compared to PB and DP. The activation status of IVB MAIT cells was similar to that of PB MAIT cells, except for a lower expression of PD-1. Both IVB MAIT cells and conventional T cells were more dominated by an effector memory phenotype compared to PB MAIT cells and T cells. IVB MAIT cells also responded more vigorously with expression of IFN-γ, granzyme B, and perforin in response to Escherichia coli stimulation compared to PB. MR1 was not expressed in syncytiotrophoblasts, but in placental villous and decidual macrophages. These data indicate that maternal MAIT cells accumulate in the intervillous space of the placenta and that they are highly armed to quickly respond if bacteria are encountered at the foetal-maternal interface.

Mucosal-Associated Invariant T Cells Display a Poor Reconstitution and Altered Phenotype after Allogeneic Hematopoietic Stem Cell Transplantation.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells which are important in the defense against certain bacteria and yeast. The reconstitution of MAIT cells after allogeneic hematopoietic stem cell transplantation (HSCT) is not known. We investigated MAIT cell phenotype and function in 17 patients devoid of relapse and severe graft-versus-host disease (GvHD) in paired samples collected 1-2, 3-6, 12, and 24 months after transplantation. Data were compared to 17 healthy controls (HC), as well as 22 patients with acute GvHD grade 2-3. The frequency of MAIT cells within CD3+ cells was approximately 10-fold lower than in HC and did not increase over the 2 years following HSCT. MAIT cells in HSCT patients displayed an elevated expression of CD69 and intracellular granzyme B and were predominantly composed of CD4/CD8 double-negative cells. The expression of PD-1 on MAIT cells was low and did not change during the observational time, whereas the CD3+CD161dim/negTCRVα7.2dim/neg cells (non-MAIT T cells) displayed a high expression early after HSCT that decreased to normal levels at 24 months. MAIT cells collected 2-6 months post-HSCT showed an impaired IFN-γ and perforin response after bacterial stimulation, but the response was restored at 24 months. Patients with acute GvHD had similar proportions of MAIT cells as patients with grade 0-1, but consisted mainly of CD8+ cells. Finally, MAIT cells were more sensitive to cyclosporine A and sirolimus than non-MAIT T cells. To conclude, MAIT cell reconstitution following HSCT is deficient compared to non-MAIT T cells and GvHD grade ≥2 is not correlated with MAIT cell frequency. MAIT cell functionality was impaired early after HSCT, but restored at 24 months post-HSCT. MAIT cells have an increased sensibility to common immunosuppressive drugs, which maybe could explain their hampered reconstitution after HSCT.

Lymphocytes in Placental Tissues: Immune Regulation and Translational Possibilities for Immunotherapy.

Immune modulation at the fetomaternal interface is crucial to ensure that the fetal allograft is not rejected. In the present review, the focus is to describe basic functions of lymphocyte populations and how they may contribute to fetomaternal immune regulation, as well as determining what proportions and effector functions of these cells are reported to be present in placental tissues in humans. Also explored is the possibility that unique cell populations at the fetomaternal interface may be targets for adoptive cell therapy. Increasing the understanding of immune modulation during pregnancy can give valuable insight into other established fields such as allogeneic hematopoietic stem cell transplantation and solid organ transplantation. In these settings, lymphocytes are key components that contribute to inflammation and rejection of either patient or donor tissues following transplantation. In contrast, an allogeneic fetus eludes rejection by the maternal immune system.

Frontline Science: Placenta-derived decidual stromal cells alter IL-2R expression and signaling in alloantigen-activated T cells.

This study investigated how stromal cells affect the IL-2 pathway in alloantigen-activated T cells. We found that decidual stromal cells (DSCs) from term placentas promoted a high production of IL-2 in cultures with alloantigen-activated T cells. The intensity of expression of cluster of differentiation 25 (CD25; IL-2Rα) on T cells was increased by DSCs, whereas the frequency and intensity of expression of the signaling subunits CD122 (IL-2Rß) and CD132 (IL-2Rγc) were reduced. Consequently, uptake of IL-2 and STAT5 phosphorylation (pSTAT5) was abrogated. DSCs also decreased the proportion of pSTAT5+ T cells in response to IL-15, which also use CD122 for signaling. Addition of DSCs to the allogeneic cultures did not increase the expression of programmed death 1 (PD-1) or CD95, indicating that they did not promote T cell exhaustion. However, exogenous recombinant (r)IL-2 in similar concentrations in the same setting increased the expression of CD95 and down-regulated CD122 in T cells. The antiproliferative effect of sirolimus (SRL) and cyclosporine A (CsA), which target the IL-2 signaling pathway, was diminished by DSCs in vitro. To conclude, DSCs affect IL-2 production and IL-2R expression and signaling, which may contribute to the stromal cell-mediated immune modulation and phenotype shift seen in activated T cells. Altered proliferation in cultures when combining DSCs and SRL or CsA may be of clinical importance, as stromal cells are used in trials for acute inflammation and are often used in combination with conventional immunosuppressive therapies.

Immunogenicity of decidual stromal cells in an epidermolysis bullosa patient and in allogeneic hematopoietic stem cell transplantation patients.

Allogeneic mesenchymal stromal cells (MSCs) are widely used in regenerative medicine, but little is known about their immunogenicity. In this study, we monitored the therapeutic and immunogenic effects of decidual stromal cells (DSCs) from term placentas when used as a therapy for generalized severe junctional epidermolysis bullosa (JEB) (previously termed Herlitz JEB), a lethal condition caused by the lack of functional laminin-332. An 11-month-old JEB patient was treated with five infusions of allogeneic DSCs within a 3-month period. Amniotic membranes (AMs) were applied to severe wounds. After the treatment, wounds started to heal in the middle of the blisters, but the improvements were transient. After two infusions of DSCs, the JEB patient had developed multispecific anti-HLA class-I antibodies. No antibodies to laminin-332 were detected, but the patient had high levels of anti-bovine serum albumin antibodies, which could bind to DSCs. Peripheral blood mononuclear cells (PBMCs) from the patient had a higher proliferative response to DSCs than to third-party PBMCs, which contrasts with the pattern observed in healthy donors. Human DSCs and MSCs induced similar xenoreactivity in mice. Two of 16 allogeneic stem cell-transplanted patients, treated with DSCs for graft-versus-host disease or hemorrhagic cystitis, showed a positive flow cytometric crossmatch test. One patient had anti-HLA antibodies before DSC infusion, whereas the other had no anti-HLA antibodies at any time. AM and DSC infusions may have improved the healing process in the JEB patient, but DSCs appeared to induce anti-HLA antibodies. The risk of alloimmunization by DSCs seems to be low in immunocompromised patients.

Treatment of severe chronic graft-versus-host disease with decidual stromal cells and tracing with (111)indium radiolabeling.

Decidual stromal cells (DSCs) isolated from fetal membranes of term placentas are easily expanded and are highly immunosuppressive in vitro. These cells express high levels of integrins that are of importance in homing to inflamed tissues. In this study, we investigated DSCs as a cellular therapy for chronic graft-versus-host disease (cGvHD), a severe complication after allogeneic hematopoietic stem cell transplantation. Subsequent to transplantation, three patients developed severe extensive cGvHD and were treated with DSCs (1-2.8 × 10(6) cells/kg). One-third of the DSCs administered to two patients were labeled with (111)Indium, and the in vivo distribution was tracked for 48 h. The (111)In-labeled DSCs were initially located in the lungs, followed by dissemination to the liver and spleen. The DSCs induced a partial response in two of the patients. Blood samples from the patients were extensively evaluated by flow cytometry, luminex, and enzyme-linked immunosorbent assay. The nonresponder had the highest proportion of T-cells with Th17 and Th2 phenotypes and the highest median plasma concentrations of IL-17 and IL-4. The same patient also had high frequencies of HLA-DR(+) T-cells and regulatory T-cells. To conclude, DSCs are safe to infuse with no adverse effects. We determined how stromal cells are distributed in vivo after infusion in a cGvHD setting. The methods established for analysis of blood samples will be useful in determining the effect of DSCs in a study comprising a larger patient material. This pilot study may provide a basis for further controlled investigations with DSCs in a clinical setting.

Low CD34 dose is associated with poor survival after reduced-intensity conditioning allogeneic transplantation for acute myeloid leukemia and myelodysplastic syndrome.

Reduced-intensity conditioning/nonmyeloablative conditioning regimens are increasingly used in allogeneic hematopoietic cell transplantation (HCT). Reports have shown CD34(+) dose to be important for transplantation outcome using myeloablative conditioning. The role of CD34(+) dose of peripheral blood progenitor cells (PBPC) has not been previously analyzed in a large population undergoing reduced-intensity conditioning/nonmyeloablative HCT. We studied 1054 patients, ages 45 to 75 years, with acute myeloid leukemia or myelodysplastic syndrome who underwent transplantation between 2002 and 2011. Results of multivariate analysis showed that PBPC from HLA-matched siblings containing <4 × 10(6) CD34(+)/kg was associated with higher nonrelapse mortality (hazard ratio [HR], 2.03; P = .001), overall mortality (HR, 1.48; P = .008), and lower neutrophil (odds ratio [OR], .76; P = .03) and platelet (OR, .76; P = .03) recovery. PBPC from unrelated donors with CD34(+) dose < 6 × 10(6) CD34(+)/kg was also associated with higher nonrelapse (HR, 1.38; P = .02) and overall mortality (HR, 1.20; P = .05). In contrast to reports after myeloablative HCT, CD34(+) dose did not affect relapse or graft-versus-host disease with either donor type. An upper cell dose limit was not associated with adverse outcomes. These data suggest that PBPC CD34(+) doses >4 × 10(6) CD34(+)/kg and >6 × 10(6) CD34(+)/kg are optimal for HLA-matched sibling and unrelated donor HCT, respectively.

Capture and separation of biomolecules using magnetic beads in a simple microfluidic channel without an external flow device.

The use of microfluidic devices and magnetic beads for applications in biotechnology has been extensively explored over the past decade. Many elaborate microfluidic chips have been used in efficient systems for biological assays. However most fail to achieve the ideal point of care (POC) status, as they require larger conventional external devices in conjunction with the microchip. This paper presents a simple technique to capture and separate biomolecules using magnetic bead movement on a microchip without the use of an external flow device. This microchip consisted of two well reservoirs (W1 and W2) connected via a tapered microchannel. Beads were dragged through the microchannel between the two wells at an equivalent speed to a permanent magnet that moved alongside the microchip. More than 95% of beads were transferred from W1 to W2 within 2 min at an average velocity of 0.7 mm s(-1). Enzymatic reactions were employed to test our microchip. Specifically, three assays were performed using the streptavidin coated magnetic beads as a solid support to capture and transfer biomolecules: (1) non-specific adsorption of the substrate, 6-8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), (2) capture of the enzyme, biotinylated alkaline phosphatase (AP), and (3) separation of AP from DiFMUP. Our non-specific adsorption assay indicated that the microchip was capable of transferring the beads with less than 0.002% carryover of DiFMUP. Our capture assay indicated efficient capture and transfer of AP with beads to W2 containing DiFMUP, where the transferred AP converted 100% of DiFMUP to DiFMU within 15 minutes. Our separation assay showed effective separation of AP from DiFMUP and elucidated the binding capacity of the beads for AP. The leftover unbound AP in W1 converted 100% of DiFMUP within 10 minutes and samples with less than the full bead capacity of AP (i.e. all AP was transferred) did not convert any of the DiFMUP. The immobilization of AP on the bead surface resulted in 32% reduced enzymatic speed compared to that of free AP in solution, as a result of altered protein conformation and/or steric hindrance of the catalytic site. Overall, this microfluidic platform was established as a simple, efficient and effective approach for separating biomolecules without any flow apparatus.