Healthy Bone Marrow Donors Research Articles

Baseline lncRNA PCAT1 high expression and its longitude increment during induction therapy predict worse prognosis in multiple myeloma patients.

BackgroundLong noncoding RNA PCAT1 (lnc‐PCAT1) involves in the proliferation and drug sensitivity of multiple myeloma (MM), while its prognostic role in MM patients is still obscure. This study aimed to explore the association of lnc‐PCAT1 with MM risk, clinical characteristics, treatment response, progression‐free survival (PFS), and overall survival (OS).MethodsA total of 83 symptomatic MM patients were enrolled in this study. Additionally, 30 healthy bone marrow donors as health controls were also recruited. Bone marrow plasma cell samples of MM patients and health donors were collected. Lnc‐PCAT1 in bone marrow plasma cells was detected by reverse transcription‐quantitative polymerase chain reaction.ResultsLnc‐PCAT1 was increased in MM patients than in health donors (p < 0.001), and receiver operating characteristic (ROC) curve showed that lnc‐PCAT1 had excellent capability of discriminating MM patients from health donors (area under curve: 0.932, 95% confidence interval: 0.889–0.976). In MM patients, lnc‐PCAT1 was correlated with bone lesion (p = 0.024), higher β2‐MG (p = 0.005), LDH (p = 0.037), and presence of Del (17p) (p = 0.029). Lnc‐PCAT1 was also associated with poor ISS stage (p = 0.013) and R‐ISS stage (p = 0.005). Besides, lnc‐PCAT1 was reduced after treatment (p < 0.001); meanwhile, lnc‐PCAT1 before treatment was correlated with lower CR (p = 0.046) but not ORR (p = 0.185). Additionally, lnc‐PCAT1 after treatment was associated with lower CR (p = 0.003) and ORR (p = 0.010). Furthermore, baseline Inc‐PCAT1 high and Inc‐PCAT1 increase after treatment were correlated with worse PFS and OS (all p < 0.05).ConclusionLnc‐PCAT1 dysregulation serves as a biomarker for diagnosis and prognosis for MM.

Sirtuin 2 knockdown inhibits cell proliferation and RAS/ERK signaling, and promotes cell apoptosis and cell cycle arrest in multiple myeloma

The present study aimed to explore the regulatory role of sirtuin 2 (SIRT2) in malignant progression of multiple myeloma (MM) and the potential associated signaling pathways. In total, 30 patients with MM and 15 healthy bone marrow donors were enrolled in the current study and their bone marrow samples were collected to isolate the plasma cells. The expression levels of SIRT2 were detected in MM cell lines (KMS-28BM, U266, RPMI-8226 and NCI-H929) and normal plasma cells (collected from healthy bone marrow donors as the control) via reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis. SIRT2 knockdown was established by transfecting two MM cell lines (RPMI-8226 and NCI-H929 cells) with short hairpin RNA-SIRT2 recombinant plasmid; the control group was transfected with a control recombinant plasmid. Subsequently, the effect of SIRT2 knockdown on MM cell proliferation, apoptosis, cell cycle progression and RAS/ERK signaling was investigated via Cell Counting Kit-8, flow cytometry, RT-qPCR and western blot assays, respectively. The mRNA and protein expression levels of SIRT2 were increased in U266 (P<0.001), KMS-28BM (P<0.001), RPMI-8226 (P<0.001) and NCI-H929 (P<0.001) cells compared with those in the control cells. In NCI-H929 and RPMI-8226 cells, cell proliferation was decreased 48 h (P<0.05) and 72 h (P<0.05) after SIRT2 knockdown. Furthermore, the cell apoptotic rate was elevated 48 h after SIRT2 knockdown (P<0.01). In addition, the percentage of cells at the G0/G1 phase was increased (P<0.01), whereas the percentage of cells at the S phase was reduced (P<0.01) 48 h after SIRT2 knockdown. The expression levels of HRAS and phosphorylated-ERK were also reduced 48 h after SIRT2 knockdown. In conclusion, SIRT2 was highly expressed in MM cell lines, and knockdown of SIRT2 inhibited MM cell proliferation, inactivated the RAS/ERK signaling pathway, and promoted cell apoptosis and cell cycle arrest.

Novel myeloperoxidase-derived HLA-A2-restricted peptides as therapeutic targets against myeloid leukemia

Background aimsHuman myeloperoxidase has been shown to be overexpressed in many types of leukemia, such as chronic myeloid leukemia, acute myeloid leukemia and myelodysplastic syndrome. The authors identified two myeloperoxidase-derived HLA-A2-restricted peptides, MY4 and MY8, as novel leukemia-associated antigens. MethodsEx vivo-elicited MY4- and MY8-specific cytotoxic T lymphocytes were generated, and tested for leukemia cell lysis in vitro and in NOD/SCID AML xenograft model. ResultsThese MY4- and MY8-specific cytotoxic T lymphocytes killed leukemic blasts while sparing healthy donor bone marrow cells. In addition, co-injection of MY4- and MY8-specific cytotoxic T lymphocytes into nonobese diabetic/severe combined immunodeficiency mice with acute myeloid leukemia drastically reduced tumor burden in vivo. The authors also found that MY4- and MY8-specific T cells could be detected in the peripheral blood mononuclear cells of allogeneic stem cell transplant recipients. ConclusionsThese antigen-specific T cells were significantly increased in blood samples from patients compared with healthy donors, suggesting that both MY4 and MY8 are immunogenic and that MY4- and MY8-specific cytotoxic T lymphocytes may play a role in reducing leukemia in vivo. Thus, the discovery of MY4 and MY8 as novel leukemia-associated antigens paves the way for targeting these antigens in immunotherapy against myeloid leukemia.

PD-1H (VISTA) Induces Immune Evasion in Acute Myeloid Leukemia

Despite progress in understanding the molecular pathogenesis in acute myeloid leukemia (AML), the disease remains highly fatal with limited therapeutic options. There is a need for novel approaches for management of AML that can overcome resistant disease. Immune checkpoint inhibitor therapy has emerged as a successful treatment strategy in several solid tumors and refractory Hodgkin lymphoma. However, preliminary data from clinical trials to date indicate that anti-PD-1 antibodies have limited single-agent activity in AML. This suggests that co-inhibitory molecules other than PD-1 may induce immune evasion in AML. Programmed Death-1 Homolog (PD-1H, VISTA) is a novel co-inhibitory molecule that induces immune evasion in solid tumors. Interestingly, The Cancer Genome Atlas (TCGA) and the Cancer Cell Line Encyclopedia database both reveal that PD-1H is over-expressed in AML at the transcript level. While we found that myeloid subsets and T cells have the expression of PD-1H in bone marrow of healthy donors, we demonstrated that PD-1H protein is highly expressed in bone marrow of 8 (of 10) human AML donors using immunohistochemistry. To determine whether the high expression of PD-1H in AML bone marrow is associated with the expression of PD-1H in AML blasts, flow cytometric analyses were performed in bone marrow aspirate of human CD34+ AML donors. Mean Fluorescent Intensity (MFI) of CD34+ AML blasts was significantly higher when compared to normal CD34+ progenitor cells (MFI: 257±82 (AML CD34+ blasts) vs 10±4 (normal CD34+ progenitor cells), N=6, p<0.05). To test our hypothesis that PD-1H induces immune evasion in AML, we transplanted PD-1H-expressing murine myeloid leukemia cells (C1498) into syngeneic PD-1H knockout (KO) or wild type (WT) littermate mice. Whole body bioluminescence (unit: radiance) was used to determine the level of luciferase activity reflecting in vivo growth of luciferase expressing C1498 cells and survival of recipient PD-1H KO mice was compared to WT controls. We observed that murine AML cell growth in vivo was diminished in PD-1H KO mice (mean radiance in one representative experiment: 2.6x106 (KO) vs 4.2x107 (WT) on day 26, N=5, p<0.05) and survival of PD-1H KO mice was improved compared to WT controls (median survival: 35 days (KO) vs 28 days (WT), N=5, p<0.05). In addition, PD-1H antibody (Ab) decreased AML cell growth in vivo in KO mice (mean radiance in one representative experiment: 2.4x105 (KO + PD-1H Ab) vs 2.6x106 (KO + Isotype control) on day 26, N=5, p<0.05) and extended survival in C1498-bearing PD-1H KO mice (median survival: 45 days (KO + PD-1H Ab) vs 35 days (KO + Isotype), N=5, p<0.05). These data suggest that PD-1H on both host cells and AML cells induces immune evasion in AML. Based on prior preclinical models, we further hypothesized that immunogenicity of AML can be enhanced by epigenetic modulation, that can potentiate anti-leukemic effect of PD-1H blockade. We found that DNA methyltransferase inhibition (DNMTi) by 5-aza-2'-deoxycytidine (decitabine) increased T cell infiltration in subcutaneously injected murine AML (C1498) tumor (CD3+ cells/tumor cells: 33±5% (DNMTi) vs 10±3% (PBS control), N=3, p<0.05) using flow cytometry and immunohistochemistry. Importantly, DNMTi potentiated the anti-leukemic effect of the PD-1H blockade (mean radiance in one representative experiment: 9.9x108 (PBS + WT) vs 1.2x108 (PBS + KO) vs 3.6x105 (decitabine + WT) vs 6.9x104 (decitabine + KO) on day 21; 5.9x109 (PBS + WT) vs 3.5x108 (PBS + KO) vs 1.8x106 (decitabine + WT) vs 1.3x105 (decitabine + KO) on day 24, N=7, p<0.05) and significantly prolonged survival in PD-1H KO mice (median survival: 28 days (WT) vs 35 days (KO) vs 39 days (DNMTi + WT) vs 60 days (DNMTi + KO), p<0.05). Together, our data suggest that PD-1H is highly expressed in human AML and can induce immune evasion in a murine model of AML (C1498). DNMTi has potential synergistic effects on PD-1H blockade and combining these two can be an important immunotherapeutic approach in AML. DisclosuresZeidan:Otsuka: Consultancy; AbbVie, Otsuka, Pfizer, Gilead, Celgene, Ariad, Incyte: Consultancy, Honoraria; Takeda: Speakers Bureau. Chen:Boehringer Ingelheim, Pfizer, NextCure: Research Funding.

Dysregulation of the ASPP Protein Family (Apoptosis Stimulating Proteins of TP53) Is a Common Feature in High-Risk Acute Myeloid Leukemia (AML)

Inactivation of the TP53 pathway is a frequent event in human cancers promoting tumorigenesis and resistance to chemotherapy. Inactivating TP53 mutations are uncommon in non-complex karyotype leukemias suggesting that the TP53 pathway must be inactivated by other mechanisms. ASPP proteins are a family of TP53-binding proteins consisting of three members: pro-apoptotic ASPP1 and ASPP2 and apoptosis-inhibiting iASPP. ASPPs control induction of apoptosis via direct interactions with TP53 but also other key proteins such as BCL-2/BXL-xl.We here reveal universal dysregulation of ASPPs in AML correlating with chemotherapy failure and provide a novel therapeutic target.mRNA and protein expression levels of ASPP1, ASPP2 and iASPP were determined in freshly isolated native patient samples (n=120) as well as healthy bone marrow donors (n=34). To mimick direct implications of the specific ASPP protein in response to chemotherapy, ASPPs were stably silenced in leukemia cell lines (MOLM14, Jurkat, HL60, NB4 or OCI-3), native patient blasts and bone marrow donor samples using a retroviral shRNA approach. Sensitivity towards standard chemotherapeutics was assessed using XTT viability and annexin V-based apoptosis assays. Transfection with an empty vector served as a negative control.Interestingly, all family members, ASPP1 (p=0.001), ASPP2 (p=0.02) and iASPP (p=0.001) were found to be significantly altered in AML compared to healthy donors. Subgroup analyses revealed association with high-risk features and failure towards induction chemotherapy. Importantly, dysregulation was not exclusive and may affect several family members - which was associated with dismal outcome.Retroviral ASPP1/2 -interference lead to perturbed proliferation capacities (up to 3-fold increase) and attenuated apoptosis in response to chemotherapy in all tested models. Vice versa, inhibition of iASPP resulted in attenuated proliferation and an increase in apoptosis.For ASPP1/2, we demonstrate promoter methylation and restoration of protein expression upon treatment with aza-nucleosides. Additional BCL-2 inhibition (using venetoclax) consequently resulted in superadditive, proapoptotic efficacy compared to the knockdown cell strains.To summarize, dysfunctional regulation of ASPP proteins is a common feature in high-risk leukemias associated with treatment failure. Restoring pro-apoptotic ASPP1/2 function using hypomethylating agents and combination with proapoptotic agents such as venetoclax may overcome therapy resistance in these settings. DisclosuresNo relevant conflicts of interest to declare.

Blocking CD70/CD27 Signaling in Combination with Hypomethylating Agents Eradicates Human CD34+ AML Stem and Progenitor Cells in Vitro and In Vivo

Leukemia stem cells (LSCs) are the origin of acute myeloid leukemia (AML) and are resistant to standard therapeutic regimens resulting in frequent relapse of the disease and poor prognosis especially for AML patients not fit for intensive chemotherapy. Consequently, LSCs represent a major obstacle for AML therapy. We recently identified the interaction of the TNF ligand CD70 and its receptor CD27 on LSCs as a promising therapeutic strategy to target LSCs.In this study, we demonstrate for the first time that treatment with hypomethylating agents (HMA, azacitidine or decitabine) significantly up-regulates CD70 mRNA and protein expression on primary CD34+ stem/progenitor cells from newly diagnosed AML patients independent of the cytogenetic/molecular risk category in vitro (mRNA: 4.3±1.6 fold increase, FACS: 4.0±0.9 fold increase vs. vehicle treatment, n=15). Similarly, CD34+ stem/progenitor cells from AML patients after 5 days decitabine administration (20mg/kg by continuous intravenous infusion over 1 hour repeated daily) exhibited a significant increase in CD70 expression as analyzed by flow-cytometry (6.8±1.1 fold increase vs. diagnostic sample, n=6). In contrast, CD70 expression of CD34+ stem/progenitor cells from healthy donor bone marrow (n=3) was not affected by the treatment.Consequently, co-treatment of CD34+ stem/progenitor cells from AML patients of each risk group (n=3) with the HMA decitabine and a blocking αCD70 monoclonal antibody strongly reduced colony-forming and re-plating capacity in vitro compared to single agent treatment. To confirm and extend our findings towards disease-initiating CD34+ AML LSCs in vivo, we performed patient-derived AML xenografts (PDX) from one patient with intermediate and one patient with adverse cytogenetic risk profile. Combining decitabine treatment with CD70 blockade significantly reduced AML engraftment in blood, spleen and BM compared to controls. Moreover, co-treatment significantly reduced frequency and absolute numbers of CD34+CD38- and CD34+CD38-CD45RA- AML LSCs in the BM as analyzed phenotypically by FACS and eliminated human LSCs as analyzed in limiting dilution PDX experiments compared to single treatments. Importantly, CD34+ hematopoietic stem/progenitor cells from healthy human bone marrow (n=3) were only marginally affected by the co-treatment.In summary, combining HMAs with blocking CD70/CD27 signaling could represent a novel strategy to eradicate human LSCs. DisclosuresVan Rompaey:argenX: Employment. Leupin:argenX: Employment. De Haard:argenX: Employment.

Checkpoint Expression By Acute Myeloid Leukemia (AML) and the Immune Microenvironment Suppresses Adaptive Immunity

Abstract Introduction AML suppresses immune responses directly by expressing cytokines and immune checkpoint ligands as well as indirectly, by recruiting suppressive immune cells like regulatory T cells (Tregs). Prior studies have looked at a small number of immune checkpoint receptors and ligands on limited subsets of T cells and AML blasts, respectively. The frequency of circulating Tregs has been associated with induction failure in AML (Szczepanski et al CCR 2009) and the coexpression of PD1 and TIM3 on CD8 T cells has been associated with immune exhaustion (Zhou et al Blood 2011) and relapse following allogeneic stem cell transplant (Kong et al Blood Can J). Recent clinical trials have demonstrated encouraging response rates and durable responses in patients with relapsed AML treated with PD-1 based therapies (Daver et al EHA 2017) and following allogeneic stem cell transplantation in relapsed AML with CTLA-4 based therapies (Davids et al NEJM 2016). Better defining the immune checkpoint landscape of human AML will facilitate the rational selection of immune checkpoint targets and combination approaches for future clinical trials. Methods Multi-parameter flow-cytometry assay were performed by Immunotherapy Platform on bone marrow (BM) aspirates from 107 patients with AML treated at the MD Anderson Cancer Center. We evaluated the expression of inhibitory (PD1, CTLA4, LAG3, TIM3) and stimulatory receptors (GITR, OX40, 41BB, ICOS) on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells and the expression of their ligands (41BBL, B7-1, B7-2, ICOSL, PDL1, PDL2 and OX40L) on AML blasts. Eight healthy donor BMs were used as controls. We correlated the expression of these markers on T cell subsets and blasts, with age, karyotype and a baseline next generation sequencing for 28 myeloid-associated genes including P53, DNA methylation proteins (DNMT3a, IDH1, IDH2, TET2) and FLT3. Results BM aspirates from AML patients demonstrated significantly higher frequency of CD45 gated CD3+ total T cell infiltrate compared to healthy donors. The proportion of CD8+ T cells expressing PD1 was higher in patients with relapsed (32.8%) and with newly diagnosed AML (26.4%) compared to healthy donors (18.5%) (P Compared with healthy donors, patients with relapsed and newly diagnosed AML had significantly higher percentage of CD8+ T cells co-expressing the inhibitory markers PD1 with TIM3 (P=0.02) and PD1 with LAG3 (P We attempted to identify potential relationships between molecular characteristics and immune checkpoint expression and found that BM blasts in patients with adverse karyotype more frequently expressed PD-L1 as compared to patients with non-adverse karyotype (17.5% vs 8.8%, P=0.03) (Figure 1D). Conclusions Increased Treg and PD1+ CD8 T cell infiltration in AML and the increased PD1+TIM3+ and PD1+LAG3+ CD8 T cells in the BMs of patients with AML, point to worsening exhaustion of an adaptive immune response. The correlation between increased Treg infiltration, increased PD1+CD8 cells and increased PD-L1 expression on blasts in the BM indicates immune suppression at multiple levels. The association between adverse cytogenetics and higher PD-L1 expression on BM blasts further suggests links between AML biology and immune suppression. The dual contributions by T cells and blasts to immune suppression indicate that these pathways may play an important role in AML survival and therefore patients may benefit from checkpoint antibody therapy. Download : Download high-res image (123KB) Download : Download full-size image Figure 1 . Disclosures Cortes: ImmunoGen: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding; Sun Pharma: Research Funding; Teva: Research Funding. Allison: Merck: Patents & Royalties: Owns patent licensed to Merck; BMS: Consultancy, Patents & Royalties; Jounce: Consultancy, Patents & Royalties: Owns patent licensed to Jounce; Jounce: Consultancy, Other: Owns stock; Kite Pharma: Consultancy, Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Constellation: Consultancy, Other: stock; GSK: Consultancy; Astra Zeneca: Consultancy; Amgen: Consultancy; EMD Serono: Consultancy; Astellas: Consultancy. Kantarjian: Delta-Fly Pharma: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Daver: Karyopharm: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Immunogen: Research Funding; Incyte Corporation: Honoraria, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Jazz: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Kiromic: Research Funding; Daiichi-Sankyo: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding.

Comparison of Outcomes after Allogeneic Hematopoietic Stem Cell Transplantation of G-CSF-Stimulated Bone Marrow Versus Non-Stimulated Bone Marrow Versus Peripheral Blood

IntroductionThe use of granulocyte colony-stimulated factor (G-CSF) mobilized peripheral blood stem cells (PBSC) has largely replaced harvested bone marrow (BM) for allogeneic stem cell transplantation. Numerous studies have shown faster neutrophil and platelet engraftment with G-CSF mobilized PBSC, leading to shorter hospitalizations; however PBSC transplantations have been associated to a higher incidence of chronic graft-versus host disease (cGVHD).Since the late 1990´s, harvested BM of healthy donors has been mobilized with G-CSF. This method increases the number of progenitor cells (CD34+) harvested, which leads to faster engraftment than with unstimulated BM (UBM).There are no reports comparing the incidence of acute and chronic GVHD with UBM, MBM, and PBSC used to treat diverse benign and malignant hematologic disorders. In this retrospective study, we compare the transplant outcomes of 160 patients from a single institution.MethodsThe charts of patients who received allogeneic HSCT between the February 2011 and September 2016 at our institution were retrospectively reviewed per an IRB approved protocol.Patients were treated according to current guidelines based on their primary diagnosis and functional status. Donors received Filgrastim (10 mcg/kg/day) for 5 doses prior to harvest. All patients received GVHD prophylaxis with methotrexate (on days +1, 3, 6, 11) and tacrolimus (from day -2 until 4 to 6 months after transplantation).Statistical analyses were performed using Minitab 17 Statistical Software 2010. A preliminary analysis was completed using Stepwise Regression/ Analyses of variance to determine any relevant covariates. ANOVA and chi-square tests were used to examine group differences in demographic and clinical characteristics or time to engraftment.ResultsTwenty-nine patients (18.1%) received MBM, 36 received UBM (22.5%), and 95 received PBSC (59.3%). There were no statistically significant differences in patient sex, primary diagnosis, donor characteristics, or disease status between groups. Statically significant differences among groups in patient age were observed (p=0.032, PBSC>UBM>MBM).Engrafment: MBM was found to have a significantly quicker engraftment of the ANC (13 days), when compared to PBSC (17 days) and UBM (19 days; p=0.01). There were no significant differences in time to engraftment of platelets, between groups.Acute GVHD: MBM was found to be associated with a significantly higher incidence of aGVHD (72%; p=0.025) versus PBSC (45%) and UBM (44%). Odds ratio's indicate that it may be up to nine times more likely and eight times more likely for MBM to develop aGVHD, when compared to UBM (95% CI: 1.15, 9.35)Chronic GVHD: Patient's treated with PBSC were found to have a higher incidence of cGVHD, when compared to MBM (45%) and UBM (39%); however these findings were not statistically significant (p=0.145) but they showed a trend to significance.Discussion and conclusionHLA-matched donors as well as related donors have been associated with lower incidence of GVHD. In our study, 100% of MBM were from related donors, 72% of UBM were from unrelated donors, and 53% of the PBSC donors were unrelated. There was a higher incidence of acute and chronic GVHD in MBM, even though they were from HLA-matched related donors; the results were statistically significant for aGVHD and there was a clear trend on cGVHD. Additionally, despite having increased incidence of acute and chronic GVHD, patients who received MBM had better overall survival; this is likely secondary to graft versus leukemia. The main limitations of the study are the sample size and that it is possible that some of the patients have not developed cGVHD yet, therefore it is necessary to obtain data from a larger cohort and with a longer follow up, additionally, since this is a retrospective study access to grade and stage of GVHD is limited for some patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

MiRNA Profiling in Pediatric T-ALL with Use of Next-Generation Sequencing: Focus on T-ALL Pathobiology and Heterogeneity

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is characterized by genetic, immunophenotypic, and clinical heterogeneity. miRNAs are small non-coding RNAs involved in post-transcriptional silencing of gene expression, thus implicated in shaping the phenotype of cells, in health and disease. Aberrantly expressed miRNAs are implicated in haematological malignancies as either oncogenic or tumor suppressor miRNAs, collectively termed oncomiRs (miRNAs related to oncogenesis). Several miRNAs have been related to leukemia progression or treatment response. The profile of miRNA expression remains to be elucidated in pediatric T-ALL.Next-generation sequencing (NGS) of the entire repertoire of expressed miRNAs (miRNA-seq) might aid the understanding of T-ALL biology and heterogeneity, providing novel candidate oncogenic/tumor suppressor miRNAs and miRNAs which might potentially serve as prognostic or classification markers in T-ALL.AimsWe aimed to characterize miRNA expression profile in pediatric T-ALL with the focus on the identification of miRNA expression profiles reflecting T-ALL heterogeneity. We aimed to assess the potential application of NGS-based miRNA profiling for the classification of pediatric T-ALL. We also aimed to identify novel candidate oncomiRs implicated in T-ALL pathobiology (potential oncogenic and tumor suppressor miRNAs).MethodsStudy group consisted of 34 pediatric T-ALL patients and 5 age-related healthy bone marrow donors. Total RNA, including small RNA fraction, was isolated using miRCURY RNA Isolation Kit-Cell & Plant (Exiqon) from CD3+ cells selected immunomagnetically by negative selection with use of BD IMag Human T Lymphocyte Enrichment Set-DM (BD Biosciences). The libraries were prepared using the NEBNext Small RNA Library preparation kit (New England Biolabs). miRNA-seq was performed using NextSeq500 Illumina, with 10 mln reads/per sample, read length: 51 bp single-end; reference annotation: GRCh37, miRbase 20 (NGS Service Exiqon).ResultsWe showed that miRNA expression profile is discriminative between pediatric T-ALL and healthy bone marrow controls. We identified 23 miRNAs overexpressed in T-ALL vs. controls (including known and candidate novel oncogenic miRNAs in T-ALL). Among these are miRNAs with known mRNA target genes implicated in T-ALL pathogenesis, including: miR-20b-5p, miR-363-3p, miR-128-3p, miR-181b-5p, miR-181a-5p. We identified 38 underexpressed miRNAs (potential tumor suppressors). Among these are miRNAs with known T-ALL-implicated targets: miR-145-5p, miR-143-3p, miR-27a-5p, miR-24-3p, miR-10b-5p. We showed that miRNA expression profile is also discriminative between immunophenotypic subtypes of T-ALL (EGIL stages: II, III, IV), based on differential expression of 94 miRNAs.Conclusion/SummaryNGS-based miRNA profiling provides insights into the heterogeneity of pediatric T-ALL at the miRNome level. Expression profiles of miRNAs differentially expressed in patients vs. controls and in T-ALL immunophenotypic subtypes, differing in their maturation stages (EGIL stages II, III, IV) hold potential for improved classification of T-ALL. Individual differentially expressed miRNAs are candidates for: functional analysis to assess their potential oncogenic/tumor suppressor role in T-ALL, and association analysis (survival and treatment response) to assess their potential as prognostic markers in this disease.Research funded by National Science Centre, Poland grant: 2014/15/B/NZ2/03394 and National Centre of Research and Development (NCRD) grant STARTEGMED3/304586/5/NCBR/2017. DisclosuresNo relevant conflicts of interest to declare.

Isolation of Circulating Plasma Cells in Multiple Myeloma on a Novel Automated System

Abstract Multiple myeloma is a cancer of disordered plasma cells. The disease poses significant challenges for clinical management where cytogenetic analysis, such as FISH (fluorescent in situ hybridization), was commonly used for diagnosis and prognosis. Beads based enrichment of plasma cells are widely used to enhance FISH sensitivity and specificity. However conventional beads-based methods often require time-consuming sample preparation and results in low cell recovery. Here we present an easy and automated method to positively isolated CD138+ plasma cells from bone marrow samples. The method is developed on a novel designed magnetic separation system, MARS®. MARS® is matrix-free magnetic separation platform providing high purity, high recovery, and high capacity for cell isolation. Magnetic bead labeled CD138+ cells are isolated directly from bone marrow and peripheral blood without centrifugation. In the pilot study, U266 cells were spiked into peripheral blood from 0.5% to 2.0% of white blood cells frequency range. In addition, healthy donor bone marrow cell sample with 0.04% plasma cells were processed on MARS®. Plasma cells were isolated at 1mL/min flow rate followed by automated rinsing and releasing on MARS®. 48% to 80% recovery of the plasma cells were obtained. Results show our method has advantage in fast (&amp;lt;30 minutes), easy process with high recovery. Our next objective will be to validate the method on Multiple Myeloma patient specimens and compared to conventional beads-based enrichment methods. Enriched plasma cells will be analyzed by FISH to prove the enhanced sensitivity with better enrichment method.

Gene expression profiling in normal cytogenetic acute myeloid leukaemia of Malay patient

Background: Insight into recent molecular analyses of patients with acute myeloid leukaemia (AML) and a normal cytogenetic have revealed a striking heterogeneity with regard to the presence of acquired changes in gene expression. Nano String technology which is aimed to identify descriptive profiling of normal cytogenetic AML in differentially expressed genes involved in the different pathways in normal cytogenetic AML patients.&#x0D; Materials and method: Blood samples were obtained from eight at diagnosis patients with normal cytogenetics AML, two follow-up patients and two normal healthy controls prior to RNA extractions. RNA gene expression assay was performed using Nano Stringn Counter® Pan Cancer Pathway Panel (Nanostring Technologies, Seattle, Washington). Briefly, 100 ng of each total RNA sample was used as input into then Counter Pan Cancer Pathway Panel sample preparation. Data was extracted using then Counter RCC Collector and raw data output was imported into nSolver v2.6 analysis software for data analysis.&#x0D; Results: The age range was from 13-69 years, with a median age-range of 41 years. We found the most enriched up regulated genes in newly diagnosed normal cytogenetic AML enlisted MPO (7.25 log FC), FLT3 (5.02 log FC), MYCN (4.99 log FC), MYB (4.74 log FC), ITGA9 (4.48 log FC), KIT (4.41 log FC), MCM2 (3.47 log FC), RAD51 (3.40 log FC), CCNA2 (3.37 log FC) and PROM1 (3.24 log FC) which those commonly be found in AML cases. For highly expressed down regulated genes were GZMB (-5.80 log FC), IL8 (-5.79 log FC), TNFRSFSF10C (-5.03 log FC), LEF1 (-4.82 log FC), IL2RB (-4.70 log FC), IL7R (-4.44 log FC), BCL2A1 (-4.15 log FC), RASGRP1 (-4.13 log FC), IL1R2 (-4.00 log FC) and HSPA6 (-3.99 log FC).&#x0D; Discussion and conclusion: Nano String required smaller amounts of starting material, and can perform mRNA expression profiling with digital precision, therefore the results do not require further validation by another method. MPO expression was significantly associated with disease-free survival (DFS). Previous report demonstrated that the groups by the MPO expression in the intermediate cytogenetic risk group showed a significant difference in DFS (p&lt;0.001). A study by Valk et al., (2004) showed FLT3, a hematopoietic growth factor receptor, is the most common molecular abnormality in AML. The presence of such mutations in FLT3 and elevated expression of the transcription factor EVI1 confer a poor prognosis. N-MYC expression levels in AML samples from patients with favorable, intermediate, or unfavorable prognosis were compared with that in CD34+ cells from four healthy bone marrow donors. The most highly down regulated gene in newly diagnosed AML goes to Granzyme B (GZMB) which involved in cytolytic activity that showed high correlation with other transcripts expressed in activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells as well as lymphocyte activation-related gene validating it as a robust and specific metric of active cellular immunity. Increased expression of IL-8 and/or its receptors has been characterized in cancer cells, endothelial cells, infiltrating neutrophils, and tumor-associated macrophages, suggesting that IL-8 may function as a significant regulatory factor within the tumor microenvironment.&#x0D; Bangladesh Journal of Medical Science Vol.20(3) 2021 p.556-562

Abstract PR003: Single-cell profiling of acute myeloid leukemia (AML) and its microenvironment reveals a CD8 continuum and adaptable T cell plasticity in response to PD-1 blockade-based therapy

Abstract Introduction: Allogeneic hematopoietic stem cell transplantation (ASCT) remains the only curative option for patients with relapsed/refractory (R/R) AML, achieved via the grafted T cells versus leukemia effect. Since ASCT is not viable option to many AML patients, our group investigated (NCT02397720) whether T cells can be harnessed to eradicate R/R AML by combining azacitidine, a hypomethylating agent, and nivolumab, a PD-1 blocker. The tumor microenvironment (TME) factors impacting response and resistance to PD-1 blockade-based treatment in AML is unknown. Methods: We performed single cell RNA sequencing (scRNAseq) of 13,633 healthy bone marrow (BM) donor, and 113,394 BM cells, paired with &amp;gt;30,000 single cell T cell receptor (scTCR) repertoires, from 22 aspirates (8 pre- and 14 post- treatment) from 8 R/R AML patients (median age 73 years) treated with azacitidine/nivolumab. 3/8 patients were responders, while 2/8 and 3/8 had stable disease, and no response, respectively. Results: We identified marked variation in the T cell components across AML patients at pre- and post- treatment, demonstrating significant dynamic changes in CD4, CD8 and non-classical T cells populations, including mucosal associated invariant T (MAIT) cells. Among CD8 cells, we identified a unique GZMK-enriched population that was higher at pretreatment in responders compared to non-responders. Pseudotemporal trajectory analysis revealed a continuum of CD8 cell states that is intermediated by the less exhausted, GZMK-enriched CD8 population. We similarly identified GZMK to discriminate between 2 MAIT populations. Further characterization of GZMK-enriched cells revealed increased expression of the stem-like T cell transcription factor TCF7, and the T cell memory transcription factor EOMES. Pathway enrichment of the 33 overlapping genes in the GZMK-enriched CD8 and MAIT versus cytotoxic T lymphocyte (CD8 CTL) signatures demonstrated highest enrichment for pathways involved in leukocyte differentiation, calcium signaling, and cytokine production. scTCR clonotype assessment revealed more shared clonotypes with the terminally effector CD8 CTL cells following PD-1 blockade. Following treatment, novel clones represented 38.7% (39/101) of total clones, followed by contracted clones (32.6%) and expanded (28.7%) clones. However, 76.9% and 72.4% of novel and expanded clones were contributed by the responders. On the other hand, non-responders contributed only 5% and 3.4% of the novel and expanded clones, respectively. Conclusion: We identified a CD8 continuum in BMs of patients with AML. The response to combined azacitidine/nivolumab is driven by emergence of novel clones and expansion of prior clones demonstrating T cell plasticity and adaptability. Our results demonstrate that the subverted T cells can be reinvigorated via PD-1 blockade and elicit responses in AML and warrants further functional characterization of GZMK expressing lymphocytes in mediating antileukemic responses. This abstract is also being presented as PO059. Citation Format: Hussein A. Abbas, Dapeng Hao, Katarzyna Tomczak, Praveen Barrodia, Jin Seon Im, Patrick K. Reville, Gheath Al-Atrash, Jing Ning, Maomao Ding, Jairo T. Mathews, Latasha Little, Jianhua Zhang, Sreyashi Basu, Marina Konopleva, Guillermo Garcia-Manero, Michael R. Green, Padmanee Sharma, James P. Allison, Steven M. Kornblau, Kunal Rai, Linghua Wang, Naval Daver, Andrew Futreal. Single-cell profiling of acute myeloid leukemia (AML) and its microenvironment reveals a CD8 continuum and adaptable T cell plasticity in response to PD-1 blockade-based therapy [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR003.

Donor characteristics and intraoperative total nucleated cell count influence hematopoietic progenitor cell yield of healthy donor bone marrow grafts.

Bone marrow graft cell content impacts engraftment potential after allogeneic hematopoietic cell transplantation (alloHCT). Surrogates, such as intraoperative total nucleated cell count (ioTNC), are of unclear utility in predicting final graft characteristics. In addition, demographic and clinical factors may influence graft cellular profile and recipient engraftment. We retrospectively reviewed marrow harvests at our institution performed between 2009 and 2019. During this time, an ioTNC was measured after 50% of the projected final graft volume was collected. Regression models were used to assess associations between ioTNC (cells/µL) and final graft CD34+ (cells/mL), and between graft and donor characteristics and final graft CD34+ (cells/mL). Fifty-three marrow harvests and donor-recipient pairs were analyzed. Median (range) donor and recipient ages were 13 (0.7-28) years and 9 (0.2-21) years, respectively. The median ratio of donor/recipient weight was 1.225kg (range 0.31-7.13). Median total volume of harvested marrow was 15.3mL/kg (range 4.3-20.4) of donor weight and 19.4mL/kg (range 4.7-87.4) of recipient weight. Median ioTNC was 20930/µL (range 6600-44310) or 2.1×109 /mL, corresponding to median predicted final graft TNC of 3.59×108 /kg recipient weight (range 1.28-19.42×108 ). Simple linear regression between ioTNC and CD34+ cells/mL resulted in an R2 of 0.42. Least absolute shrinkage and selection operator (LASSO) regression produced a moderately predictive model consisting of ioTNC, donor age, and donor weight (adjusted R2 =0.7) of final graft CD34+ cells/mL. ioTNC and certain donor characteristics correlate moderately well with marrow product CD34+ cells/mL, potentially informing donor selection and marrow procurement strategies.

Expression and Clinical Significance of BCL2 Interacting Protein 3 Like in Multiple Myeloma.

Multiple myeloma (MM) is one of the main blood disorders threatening human health today. This study aimed to examine the expression of BCL-2/adenovirus E1B 19 kDa-interacting protein 3-like (BNIP3L) in patients with MM and explore its mechanisms in silico. Bone marrow samples (n = 36 from patients with MM and n = 12 from healthy donors) were used to conduct BNIP3L expression analysis using immunohistochemistry. Microarray or RNA sequencing data from the Sequence Read Archive, Gene Expression Omnibus, and ArrayExpress databases were used to appraise BNIP3L expression and its prognostic role in patients with MM. The co-expressed genes of BNIP3L were identified for enrichment and protein-protein interaction (PPI) analyses to determine the associated signaling pathways. Immunohistochemistry indicated that BNIP3L expression in bone marrow of patients with MM was significantly lower than that in bone marrow of healthy donors. BNIP3L mRNA expression was also significantly lower in patients with MM than in healthy donors. The overall standard mean difference (SMD) for downregulation of BNIP3L was −0.62 [−1.17, −0.06], and the area under the curve was 0.81 [0.78, 0.85] based on a total of 694 MM cases. The overall survival analysis demonstrated that BNIP3L levels could act as an independent protective indicator of MM patient survival (HR = 0.79). Moreover, 261 co-expressed genes of BNIP3L were confirmed and found to be mainly involved in the adipocytokine signaling pathway. We preliminarily proved that downregulation of BNIP3L may play an important role in the occurrence and development of MM, and the promoting cancer capacity may be related to the pathway of adipocytokine signaling pathway.

Toll-Like Receptor Expression of Mesenchymal Stem Cells Derived from Granulocyte Colony-Stimulating Factor Treated Bone Marrow Donors

Objective: The present study was planned to examine the expression of Toll-like receptors on human marrow-derived mesenchymal stem cells as a result of in-vivo exposure to granulocyte colony-stimulating factor with or without exposure of the cells to Toll-like receptors agonists.&#x0D; Materials and Methods: Toll-like receptor 2, 3, and 4 expressions of mesenchymal stem cells obtained from healthy human bone marrow donors exposed to in-vivo granulocyte colony-stimulating factor were analyzed, and granulocyte colony-stimulating factor untreated donors served as controls. Also, mesenchymal stem cells were stimulated in-vitro by Toll-like receptor agonists to observe the changes in the expression of the Toll-like receptors.&#x0D; Results: Mesenchymal stem cells obtained from both granulocyte colony-stimulating factor exposed or unexposed donors showed a low level of Toll-like receptor 2, 4 expressions by flow cytometry, whereas Toll-like receptor 3 expression was higher. Lipopolysaccharide was used as an agonist, but no significant difference was observed in the Toll-like receptor 2, 4 expressions, both in the granulocyte colony-stimulating factor exposed and unexposed groups. Stimulation of cells with Toll-like receptor 3 ligand was associated with a statistically significant decrease in Toll-like receptor 3 expressions, which was more profound in granulocyte colony-stimulating factor unexposed cells.&#x0D; Conclusion: We have shown that human bone marrow-derived culture-expanded mesenchymal stem cells express Toll-like receptor 3, whether in-vivo granulocyte colony-stimulating factor treated or untreated. Besides, the Toll-like receptor 3 agonist’s effect in lowering the expression levels was more significant in cells that were not exposed to granulocyte colony-stimulating factor. Additionally, detection of low expression of the pro-inflammatory Toll-like receptor 4 versus higher levels of Toll-like receptor 3 supports literature regarding the immunosuppressive characteristics of marrow-derived mesenchymal stem cells. Modulation of the expression of the Toll-like receptor of mesenchymal stem cells with granulocyte colony-stimulating factor or agonists may have implications in allogeneic mesenchymal stem cell therapies.

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion.

The gene CXXC5, encoding a retinoid-inducible nuclear factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome and adult acute myeloid leukemia. RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknown. Here, we evaluated the consequences of RINF silencing on cytokine-induced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells, without affecting cell viability. The phenotype induced by RINF-silencing was dependent on tumor growth factor b (TGFb) and mediated by SMAD7, a TGFb-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and myelodysplastic syndrome patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdown-dependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a TET2-anchoring platform in mouse. Collectively, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFb superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

Single cell sequencing reveals cell populations that predict primary resistance to imatinib in chronic myeloid leukemia.

The treatment of chronic myeloid leukemia (CML), a disease caused by t(9;22)(q34;q11) reciprocal translocation, has advanced largely through the use of targeted tyrosine kinase inhibitors (TKIs). To identify molecular differences that might distinguish TKI responders from non-responders, we performed single cell RNA sequencing on cells (n = 41,723 cells) obtained from the peripheral blood of four CML patients at different stages of treatment to generate single cell expression profiles. Analysis of our single cell expression profiles in conjunction with those previously obtained from the bone marrow of additional CML patients and healthy donors (total = 69,263 cells) demonstrated that imatinib treatment significantly altered leukocyte population compositions in both responders and non-responders, and affected the expression profiles of multiple cell populations, including non-neoplastic cell types. Notably, in imatinib poor-responders, patient-specific pre-treatment unique stem/progenitor cells became enriched in peripheral blood compared to the responders. These results indicate that resistance to TKIs might be intrinsic in some CML patients rather than acquired, and that non-neoplastic immune cell types may also play vital roles in dispersing the responsiveness of patients to TKIs. Furthermore, these results demonstrated the potential utility of peripheral blood as a diagnostic tool in the TKI sensitivity of CML patients.

Hematopoietic Stem Cells Are Endowed with Erythroid Signature in Beta-Thalassemia

Background Beta-thalassemia (Bthal) is a genetic disorder due to mutations in the ß-globin gene, leading to a reduced or absent production of HbA, which interferes with erythroid cell maturation and limits normal red cell production. Patients are affected by severe anemia, hepatosplenomegaly, and skeletal abnormalities due to rapid expansion of the erythroid compartment in bone marrow (BM) caused by ineffective erythropoiesis. In a classical view of hematopoiesis, the blood cell lineages arise via a hierarchical scheme starting with multipotent stem cells that become increasingly restricted in their differentiation potential through oligopotent and then unipotent progenitors. In human, novel purification strategies based on differential expression of CD49f and CD90 enrich for long-term (49f+) and short-term (49f−) repopulating hematopoietic stem cells (HSCs), with distinct cell cycle properties, but similar myeloid (My) and lymphoid (Ly) potential. In this view, it has been proposed that erythroid (Ery) and megakaryocytic (Mk) fates branch off directly from CD90-/49f− multipotent progenitors (MPPs). Recently, a new study suggested that separation between multipotent (Ery/My/Ly) long-term repopulating cells (Subset1, defined as CLEC9AhighCD34low) and cells with only My/Ly and no Ery potential (Subset2, defined as CLEC9AlowCD34high)occurs within the phenotypic HSC/MPP and CD49f+ HSCs compartment. Aims A general perturbed and stress condition is present in the thalassemic BM microenvironment. Since its impact on the hematopoietic cell subpopulations is mostly unknown, we will investigate which model of hematopoiesis/erythropoiesis occurs in Bthal. Moreover, since Beta-Thalassemia is an erythropoietic disorder, it could be considered as a disease model to study the 'erythroid branching' in the hematopoietic hierarchy. Methods We defined by immunophenotype and functional analysis the lineage commitment of most primitive HSC/MPP cells in patients affected by this pathology compared to healthy donors (HDs). Furthermore, in order to delineate the transcriptional networks governing hematopoiesis in Beta-thalassemia, RNAseq analysis was performed on sorted hematopoietic subpopulations from BM of Bthal patients and HDs. By droplet digital PCR on RNA purified from mesenchymal stromal cells of Bthal patients, we evaluated the expression levels of some niche factors involved in the regulation of hematopoiesis and erythropoiesis. Moreover, the protein levels in the BM plasma were analyzed by performing ELISA. Results Differences in the primitive compartment were observed with an increased proportion of multipotent progenitors in Bthal patients compared to HDs. The Subset1 compartment is actually endowed with an enhanced Ery potential. Focusing on progenitors (CD34+ CD38+) and using a new sorting scheme that efficiently resolved My, Ery, and Mk lineage fates, we quantified the new My (CD71-BAH1-/+) and Ery (CD71+ BAH1-/+) subsets and found a reduction of Ery subset in Bthal samples. We can hypothesize that the erythroid-enriched subsets are more prone to differentiate quickly due to the higher sensitivity to Epo stimuli or other bone marrow niche signals. Gene set enrichment analysis, perfomed on RNAseq data, showed that Bthal HSC/MPP presented negative enrichment of several pathways related to stemness and quiescence. Cellular processes involved in erythropoiesis were found altered in Bthal HSC. Moreover, some master erythroid transcription factors involved were overrepresented in Bthal across the hematopoietic cascade. We identified the niche factors which affect molecular pathways and the lineage commitment of Bthal HSCs. Summary/Conclusions Overall, these data indicate that Bthal HSCs are more cycling cells which egress from the quiescent state probably towards an erythroid differentiation, probably in response to a chronic BM stimulation. On the other hand,some evidences support our hypothesis of an 'erythroid branching' already present in the HSC pool, exacerbated by the pathophysiology of the disease. Disclosures No relevant conflicts of interest to declare.

Single-Cell Characterization of Acute Myeloid Leukemia (AML) and Its Microenvironment Identifies Signatures of Resistance to PD-1 Blockade Based Therapy

Single-Cell Characterization of Acute Myeloid Leukemia (AML) and Its Microenvironment Identifies Signatures of Resistance to PD-1 Blockade Based Therapy

Reevaluation of reference values for bone marrow differential counts in 236 healthy bone marrow donors

Despite the increasing role of molecular markers, differential counts and morphology of hematopoietic cells in the bone marrow (BM) remain essential diagnostic criteria in hematological diseases. However, the respective reference values for BM myelogram commonly used came from small series with limited numbers of healthy individuals. We evaluated the myelograms of 236 healthy individuals who underwent unrelated bone marrow donation. Health check-ups were performed 4 weeks prior to harvest. Samples for this study, taken from the first aspiration, were stained according to the standard Pappenheim method. Three experienced investigators assessed cellularity, megakaryopoiesis, and differential counts independently. The median donor age was 31 (range 18–51) years. Predonation tests did not reveal any relevant morbidity. Thirty-seven out of 42 hypocellular marrow samples were from younger donors up to 39 years. Content of megakaryocytes was normal in 210 specimens (89%). Gender and body mass index had significant impact on hematopoiesis, whereas age had not. The number of erythroblasts was higher (about 32%) and the proportion granulopoiesis slightly lower (about 50%) compared with previous studies. Differential counts showed also some differences with respect to individual maturation stages in these lines. Interrater comparisons showed greater reliability for the assignment of cells to the different hematopoietic cell lines than for single-cell diagnoses. This study largely confirms the results for cell counts in normal human bone marrow available from previous reports and provides some insights into factors that affect individual cell populations. It also reveals substantial variability among even experienced investigators in cytological diagnoses.