Anesthetic Management of Healthy Donor for Bone Marrow Harvesting

Patients with the rare pre-leukemia bone marrow failure syndrome severe congenital neutropenia (CN) have markedly reduced numbers of neutrophils in peripheral blood (<500/μl), leading to frequent infections and requiring chronic granulocyte stimulating factor (G-CSF) treatment. Approximately 7 % of CN patients carry homozygous loss-of-function mutations in the HAX1 gene. 25 % of HAX1-CN patients develop MDS or AML. The only curative therapy for CN patients with overt MDS/AML is hematopoietic stem cell transplantation with its associated risks. A clinical need for gene therapy for CN patients is imminent. Here, we describe for the first time the application of CRISPR/Cas9 gene-editing in combination with recombinant adeno associated virus 6 (rAAV6)-based delivery of the template for homology-directed repair (HDR) for the mutated HAX1 gene in primary bone marrow mononuclear CD34+ cells (HSPCs) of HAX1-CN patients. We selected HAX1 mutation p.W44X as the most frequently described mutation in HAX1-CN. We established the delivery of the chemically modified sgRNA in combination with SpCas9 V3 in primary HSPCs using electroporation. The HDR template was generated by PCR from healthy donor HSPCs and cloned into pRC6 vector for the production of high titer rAAV6 (>12x1012 viral copies per ml). Our gene-editing protocol produced on average 79,7 % (± 8,62 %) of total editing (TE) in healthy donor HSPCs (n=6). When we transduced healthy donor HSPCs with rAAV6 containing the template at MOI 105 after electroporation with CRISPR/Cas9 RNP, we achieved 38,1 % (± 1,3 %) knock-in (KI) efficiency and 82,3 % (± 8,2 %) TE (n=2). We further applied this approach to primary HSPCs from 5 CN patients harboring the p.W44X HAX1 mutation. We achieved 84,4 % (± 4,2 %) TE and 65,8 % (± 7,12 %) KI. Too proof, that our editing reintroduced HAX1 protein expression, we performed Western Blot analysis of edited cells (n=2) and were able to detect relevant amounts of HAX1 protein. To assess the effect of HAX1 correction on the neutropenic phenotype in vitro, we performed a liquid culture differentiation assay of edited HSPCs to neutrophils. HSPCs from the same patients that were edited in the AAVS1 safe harbor were used as isogenic controls. In the AAVS1 locus the editing efficiency was 76,74 % (± 17,07 %) total indels. By morphological assessment of Wright-Giemsa stained cytospins of edited cells derived on day 14 of differentiation revealed significant (p = 0,005) increases of mature neutrophils for all five edited HAX1-CN patient samples, as compared to the respective controls. This phenotype correction was also observed in flow cytometry by a significant (p = 0,011) increase of mature CD34-CD45+ CD15+CD16+ neutrophils (n=5). To investigate if the HAX1 mutation correction and reinforced expression of HAX1 protein improved the sensitivity of HSPCs to oxidative stress as described by Klein et al. 2007, we performed live-cell imaging of caspase3/7 activation. Live-cell imaging revealed a substantial reduction of H2O2-induced apoptosis in corrected HAX1-CN patients derived HSPCs (n=3). Furthermore, the corrected differentiated cells were investigated for functional hallmarks of granulocytes. We could observe that HAX1 gene-edited HSPCs showed comparable chemotaxis, phagocytosis and no defects in ROS production to isogenic control edited cells. Taken together, we established a protocol for efficient selection-free correction of HAX1 p.W44X mutation in primary HSPCs using CRISPR/Cas9 and rAVV6 HDR repair templates. Our gene-editing reintroduced HAX1 protein expression in primary HSPCs from HAX1-CN patients. Neutrophils derived from corrected cells showed functional improvements in survival to oxidative stress and general neutrophil functions. We believe that these results are enticing to be investigated further for potential clinical translation as an autologous stem cell therapy for HAX1-CN patients. Disclosures No relevant conflicts of interest to declare.

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Cinematic Rendering Following a Bone Marrow Harvesting Procedure

Radiology ◽

10.1148/radiol.2019190204 ◽

2019 ◽

Vol 291 (2) ◽

pp. 299-299

Author(s):

Hannah S. Recht ◽

Elliot K. Fishman

Keyword(s):

Bone Marrow ◽

Cinematic Rendering ◽

Bone Marrow Harvesting

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Healthy Donor Whole Bone Marrow Cells Preconditioned With Myeloma Patient Serum Support Long-Term Survival Of Primary Myeloma and Reveal Altered Microenvironmental Pathways

Blood ◽

10.1182/blood.v122.21.3118.3118 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3118-3118

Author(s):

Rakesh Bam ◽

Sathisha Upparahalli Venkateshaiah ◽

Xin Li ◽

Sharmin Khan ◽

Wen Ling ◽

...

Keyword(s):

Bone Marrow ◽

Healthy Donor ◽

Plasma Cells ◽

Healthy Adult ◽

Bone Marrow Cells ◽

Conflicts Of Interest ◽

Statistical Significance ◽

Term Survival ◽

Long Term Survival

Abstract Primary human myeloma (MM) cells do not survive in culture while current in vitro and in vivo systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animal model. The aim of the study was to determine long-term survival and interaction of primary MM plasma cells with a healthy adult human BM that include immune cells capable of functional activation. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum pooled from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such as monocytes, B and T lymphocytes and NK and normal plasma cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 20 patients were added to the BM cultures (4:1 BM:MM cell ratio) and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45low/intermediate/CD38high MM plasma cells among total number of cells. MM and BM cell viability was constantly high (>90%) in cocultures. Subsets of primary MM plasma cells, regardless of molecular risk or subtype, were survived and detected in all cases while in 14 of 20 experiments, number of MM plasma cells was increased by 58±12% (p<0.0005, n=14). MM cell proliferation following long-term coculture was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of primary MM was superior in cocultures supplemented with patient serum compared to healthy donor serum. In additional study, we stably infected IL6- or stroma-dependent MM lines, or two primary MM cell cases capable of passaging in SCID-hu mice with EGFP/luciferase construct and demonstrated increased MM cell growth in all experiments in coculture using bioluminescence analysis (statistical significance range: p<0.04 to p<0.0003). Growth of OPM2 MM line was also enhanced in coculture compared to culture alone. The coculture conditions protected OPM2 cells from dexamethasone but not bortezomib while proportion of MM cell killing by lenalidomide was enhanced compared to culture of OPM2 cells alone. To assess the effect of MM cells on BM cells in coculture, global gene expression profile was performed on BM cells cultured alone or plasma cell-depleted BM after coculture with MM cells from 4 patients. Among the top underexpressed genes we identified immunoglobulin genes related to polyclonal plasma cells, extracellular factors associated with osteoblastogenesis (e.g. MGP, IGFBP2), WNT signaling (e.g. SOX4, LRP1, LRP6) and TGFb bioavailability (e.g. FBN1, LTBP1). Top upregulated genes include immuneregulatory factors such as PROK2, LRG1, OLFM4 and IL16, and cellular markers (e.g. ARG1 expressed by MDSCs). This culture system demonstrates the ability of primary MM cells to interact with and to survive in coculture with healthy adult BM that was first cultivated by patients' serum and is appropriate for studying MM-microenvironment interaction, characterization of MM cell subpopulations capable of long term survival and targeted therapy. Disclosures: No relevant conflicts of interest to declare.

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Histone Acetyltransferase Pcaf Is Required for ATRA-Induced Granulocytic Differentiation in Acute Promyelocytic Leukemia Cells

Blood ◽

10.1182/blood.v124.21.617.617 ◽

2014 ◽

Vol 124 (21) ◽

pp. 617-617

Author(s):

Yoshitaka Sunami ◽

Marito Araki ◽

Akihiro Ito ◽

Yumi Hironaka ◽

Minoru Yoshida ◽

...

Keyword(s):

Bone Marrow ◽

Cell Differentiation ◽

Healthy Donor ◽

Mrna Level ◽

Hematologic Malignancies ◽

Primary Cells ◽

Atra Treatment ◽

Granulocytic Differentiation ◽

Mrna Induction ◽

Left And Right

Abstract Differentiation therapy with All-trans retinoic acid (ATRA) improves the treatment outcome of acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML); however, its molecular mechanism remains elusive. We have previously reported that inhibition of NAD-dependent histone deacetylase SIRT2 induces granulocytic differentiation in APL cells (PLOS ONE. 2013; 8(2): e57633), suggesting a possible involvement of protein acetylation in the differentiation of APL cells by ATRA. To assess this possibility, in the present study, we examined expression of major histone-acetyltrasnferases such as GCN5, PCAF, and CBP/p300 upon ATRA-treatment, and found that PCAF-expression was dramatically increased at mRNA (1.8 to 7 fold, Figure 1A) as well as protein levels in NB4, HL-60, and APL primary cells. Consistent with this, PCAF mRNA expression was greatly induced (10 to 40 fold) in the bone marrow of APL patients who received ATRA-containing treatment (Figure 1B). A search in the public expression database revealed that PCAF-expression in bone marrow was generally reduced in not only APL but also other acute hematologic malignancies such as AML and acute lymphoblastic leukemia (ALL) (healthy donor; n = 74, APL; n=37, AML; n=505, ALL; n=750, healthy donor vs. AML, p < 0.001, healthy donor vs. APL, p < 0.001, healthy donor vs. ALL, p < 0.001, adopted from oncomine database). Interestingly, the reduction of PCAF-expression was much more evident in acute than chronic hematologic malignancies such as chronic myeloid leukemia (CML) and myelodysplastic syndrome (MDS) (CML; n = 76, MDS; n=206), suggesting that PCAF reduction is related to a blockade of proper differentiation in malignant cells. Based on these findings we hypothesized that PCAF plays an important role in ATRA-induced APL cell differentiation. To prove this, we performed a loss-of-function assay using lentivirus vectors expressing 3 independent shRNA against PCAF or non-targeting shRNA (control) (Figure 1C). When PCAF was knocked down in HL-60 cells, these cells failed to differentiate into granulocytes with ATRA-treatment (31.9±11.9% vs. 4.9±2.2%, 5.2±0.7%, 8.6±3.7%, CD11b positive cells, control vs. 3 independent PCAF shRNA) (Figure 1C). Moreover, transduction of PCAF shRNA or non-target shRNA to primary APL cells made cells resistant to ATRA-induced granulocytic differentiation (data not shown), suggesting that PCAF is required for the ATRA-induced granulocytic differentiation in APL cells. To further investigate how PCAF promotes APL cell differentiation upon ATRA-treatment, we performed an acetylome analysis to identify a downstream molecule whose activity is regulated by PCAF through acetylation. We pulled-down acetylated proteins using anti-acetylated lysine antibody from cell extracts prepared 3 or 24 hours after either mock- or ATRA-treatment, and identified 5 proteins preferentially acetylated by ATRA-treatment including histone H3, which is a known acetylation target of PCAF. These results strongly support our hypothesis that upon ATRA-treatment, PCAF induction and subsequent acetylation of PCAF substrates promotes APL cell differentiation. More detailed understanding of PCAF-dependent APL cell differentiation may lead to the development of differentiation therapy against not only APL but also other AML. Figure 1 (A) PCAF mRNA induction in ATRA-treated NB4, HL-60, and APL primary cells. (B) PCAF induction in the bone marrow of APL patients during the ATRA-containing chemotherapy. Day 1 represents the mRNA level prior to the treatment. (C) PCAF knocked-down in HL-60 cells by 3 independent shRNA against PCAF or non-targeting shRNA (control) (Upper left). PCAF knocked-down cells lack a capacity to differentiate into granulocytes (CD11b positive cells) by ATRA-treatment (***; p<0.001, n=3) (lower left, and right). Figure 1. (A) PCAF mRNA induction in ATRA-treated NB4, HL-60, and APL primary cells. (B) PCAF induction in the bone marrow of APL patients during the ATRA-containing chemotherapy. Day 1 represents the mRNA level prior to the treatment. (C) PCAF knocked-down in HL-60 cells by 3 independent shRNA against PCAF or non-targeting shRNA (control) (Upper left). PCAF knocked-down cells lack a capacity to differentiate into granulocytes (CD11b positive cells) by ATRA-treatment (***; p<0.001, n=3) (lower left, and right). Disclosures No relevant conflicts of interest to declare.

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Enhanced VISTA Expression in a Subset of Patients with Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.4056.4056 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4056-4056 ◽

Cited By ~ 2

Author(s):

Adam Lamble ◽

Yoko Kosaka ◽

Fei Huang ◽

A Kate Sasser ◽

Homer Adams ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Myeloid Leukemia ◽

Research Funding ◽

Healthy Donor ◽

M2 Macrophages ◽

High Group ◽

Hla Dr ◽

Healthy Donors ◽

Equity Ownership

Abstract Introduction: Identification of tumor associated antigens (TAA) in acute myeloid leukemia (AML) will facilitate the use of targeted immunotherapies such as chimeric antigen receptor T cell therapy and bispecific antibodies. The characteristics of an ideal TAA include tumor specificity, high expression level, large percentage of positive tumor cells, and evidence for expression on putative cancer stem cells. V-domain Ig Suppressor of T cell Activation (VISTA) is a cell surface protein previously shown to be present on hematologic cells and inhibitory towards T cells. VISTA's role and presence within AML has yet to be defined. We performed detailed studies to characterize VISTA expression on tumor cells and mononuclear cell subsets in patients with AML. Methods: Immunophenotyping was performed on mononuclear cells from primary AML patient specimens using mass cytometry (CyTOF) by staining samples with a panel containing 34 antibodies to surface markers and cytokines (Table 1). This antibody panel was used to identify blast cells (variable immunophenotype), leukemic stem cells (LSCs) (CD34+, CD38-), T cells (CD3+), monocytes (CD33+, CD14+, CD11b+, HLA-DR+), myeloid derived suppressor cells (MDSCs) (CD33+, CD11b+, HLA-DR-), and M2 macrophages (CD11b+, CD163+). Raw median VISTA expression and percentage of cells expressing VISTA were calculated for all of these cell types. Samples were split into 2 categories based on blast VISTA expression. A sample was considered VISTA high if >10% of the blasts expressed VISTA, and VISTA low if <10% of the blasts expressed VISTA. Similar data was generated from a pool of healthy donors for comparison. Results: Fourteen bone marrow samples from patients with newly diagnosed AML and 10 from healthy donors were obtained. The average percentage of blasts expressing VISTA was 68% (range 28% - 81%) in the VISTA high group and 4.2% (range 0.3% - 6.8%) in the VISTA low group (p<0.05). VISTA expression was higher in the VISTA high group than in the VISTA low group (17.7 vs -0.1, p<0.05). The VISTA high group had a lower blast percentage in the bone marrow compared to the VISTA low group (56% vs 91%, p<0.05). The VISTA high and low groups had similar percentages of LSCs (2.5% vs 0.6%, p=0.11), but the LSCs in the VISTA high group maintained higher VISTA expression than did those in the VISTA low group (6.81 vs -0.11, p<0.05). The LSCs within the VISTA high group had similar VISTA expression to the blast population (6.81 vs 17.7, p=0.15). Within the non-blast population, VISTA expression was higher on the myeloid cell subsets than on the T cells for both the AML samples (11.5 vs 0.17, p<0.05) and the healthy donor samples (8.07 vs 0.24, p<0.05). VISTA expression was similar between the VISTA high and low groups on T cells (0.36 vs 0.02, p=0.08), monocytes (15.7 vs 19, p=0.7), MDSCs (6.3 vs 4, p=0.26), and M2 macrophages (7.4 vs 16.6, p =0.25). VISTA expression was similar between AML and healthy donor samples for T cells (0.17 vs 0.24, p=0.6), monocytes (15.7 vs 14, p=0.41), MDSCs (6.3 vs 3.6, p=0.22), and M2 macrophages (7.4 vs 6.6, p=0.19). Monocytes had the highest expression of VISTA within the healthy donor samples (p<0.05) and this difference was maintained in the AML samples. Conclusion: A subset of patients with AML has a large percentage of blasts, including LSCs, with high VISTA expression on their surface. VISTA is expressed on normal myeloid cells in the bone marrow of patients with AML, but at low levels and to a similar degree as in the marrow of healthy donors. Disclosures Huang: Janssen Research & Development, LLC: Employment, Other: I am an employee of Janssen and a stock owner . Sasser:Janssen Research & Development, LLC: Employment. Adams:Janssen Research & Development, LLC: Employment. Druker:Agios: Honoraria; Ambit BioSciences: Consultancy; ARIAD: Patents & Royalties, Research Funding; Array: Patents & Royalties; AstraZeneca: Consultancy; Blueprint Medicines: Consultancy, Equity Ownership, Other: travel, accommodations, expenses ; BMS: Research Funding; CTI: Equity Ownership; Curis: Patents & Royalties; Cylene: Consultancy, Equity Ownership; D3 Oncology Solutions: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses ; Lorus: Consultancy, Equity Ownership; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Novartis: Research Funding; Oncotide Pharmaceuticals: Research Funding; Pfizer: Patents & Royalties; Roche: Consultancy. Lind:Janssen: Research Funding; Fluidigm: Honoraria.

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Outcomes of autologous blood collection prior to bone marrow harvest for matched unrelated donors.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e18535 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e18535-e18535

Author(s):

Brian Hemendra Ramnaraign ◽

Brittany Kayla Rogers ◽

Susan P. McGorray ◽

Michele Sugrue ◽

Hemant S. Murthy ◽

...

Keyword(s):

Bone Marrow ◽

Blood Transfusion ◽

Autologous Blood ◽

Blood Collection ◽

Common Procedure ◽

Post Harvest ◽

Patient Request ◽

Unrelated Donors ◽

And Gender ◽

Bone Marrow Harvesting

e18535 Background: Autologous (auto) blood collection prior to bone marrow (BM) harvest is a common procedure however there is little data to say whether this is beneficial. Methods: This is a retrospective study evaluating the efficacy of pre-op auto blood collection in healthy unrelated donors who underwent BM harvesting at our institution between 9/2009 and 8/2017. Unrelated donors aged 18 or older who underwent their first BM harvest were included. Comparisons were made using two sample t-tests. Results: Among the 73 BM donors, 54 (74%) underwent auto blood collection resulting in 78 units collected. The cohorts with and without auto blood collected were similar in age and gender (mean age 31.0 vs 30.2; female 59% vs 41%). Those with auto blood collected donated larger volumes of marrow (mean 1395 mL vs 799 mL, p = 0.0002). Baseline hemoglobin (Hb) was similar between the cohorts (mean 14.1 g/dL vs 14.0 g/dL, p = 0.87). However, those with auto blood collected had lower pre-harvest (mean 13.1 g/dL vs 13.8 g/dL, p = 0.0430) and post-harvest Hb (mean 10.0 g/dL vs 11.3 g/dL, p = 0.0120). Of the 78 auto blood units collected, 45 units (58%) were used with 92% of women and 56% of men receiving their auto blood post-op. 33 (42%) auto blood units were discarded. Donors who were given back their auto blood were more likely to be female and have lower pre- and post-harvest Hb. Reasons for auto blood transfusion were blood availability (31%), donor post-op symptoms (i.e. hypotension, dizziness, syncope, and bleeding; 23%), intra-operative transient hypotension (26%), post-op anemia (11%) and patient request (9%). None of the patients who were transfused auto blood, or donors without auto blood collection, required allogeneic blood. Conclusions: Based on our results, collection of auto blood prior to bone marrow harvest leads to lower pre-op Hb and increases the likelihood of post-op blood transfusion. In addition, availability of auto blood can lead to over transfusion which may be detrimental to donor health. Although there may be a subset of donors who might benefit, routine auto blood collection prior to bone marrow harvesting is unnecessary and can be potentially hazardous to donors.

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