PB Samples Research Articles

Impact of blood source and component manufacturing on neurotrophin content and in vitro cell wound healing.

We evaluated neurotrophin (NF) levels and their impact on in vitro cell wound healing in eye drops from differently prepared blood sources (cord blood [CB], and peripheral blood [PB]) in the same donor, to avoid intrasubject biological variability. Twenty healthy adult donor PB samples, and twenty CB samples acquired at the time of delivery were processed to obtain serum (S), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and S retrieved from PRP after activation with Ca-gluconate (PRP-R). The levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF), and epidermal growth factor (EGF) were assessed with a Luminex xMAP (Luminex Corporation), and by using multikine kits from R&D system, and were statistically analysed in the eight different preparations. The impact of S, PRP, PPP, PRP-R from both sources on a cell line responding to NF supplementation (MIO-M1, UCL Institute of Ophthalmology, London, UK) was tested with a scratch wound assay, and analysed by IncuCyte S3 equipment. All the preparations from CB showed higher NF levels, except for BDNF where no difference was found as compared to PB. PRP showed higher NF levels with respect to S, PPP and PRP-R in this decreasing order. Younger donors in PB contributed with higher NF levels. The scratch assay showed different cell migration results, with a complete wound closure only recorded with the supplementation of CB-S, and a progressive reduction by using PRP, PRP-R, and PPP from both sources. Protocols of preparation and choice of blood source determine different NF levels in the final products. The therapeutic use of a natural neurotrophin pool from blood sources might have a clinical impact in several different settings. Efforts are needed to standardise the manufacturing and the product content in order to establish and modulate the posology of the final supplementation.

Effect of multiple keyway notches on the fracture behaviour of unirradiated Gilsocarbon graphite I: Small-scale sample sizes and FE modelling

Four-point bend (4PB) samples with seven notch configurations and three-point bend (3PB) samples with four notch configurations have been designed and tested in this study to evaluate the fracture behaviour of virgin Gilsocarbon graphite with simulated keyway notches. The effective stress concentration factor for Gilsocarbon graphite with simulated keyway profile notches was estimated to be around 1.2, which was much smaller compared with the FE modelled theoretical stress concentration factor of ∼ 3.4. In most times, failure occurred at the corner which was under the maximum applied principal stress. However, the large variability in the strength of Gilsocarbon graphite may result in crack initiation location not associated with a largest stress intensification, such as the flat regions of the keyway notches and the notch corners that are not under the maximum applied stress.The number of notches within the sample, combinations of notches, notch distance ratios and a distance between the edges of neighbouring notches were found to have little effect on the nominal net-section fracture strength (nominal strength) of 4PB samples (∼22 MPa). While, the smaller distance between the edges of neighbouring notches (Ln ∼ 8 mm) in 3PB sample configurations T1 and T3 resulted in a shielding effect at the inner notch corners, which in turn resulted in a slightly larger nominal strength at T1 and T3 configurations (∼24 MPa) than that of T2 and T4 configurations (∼22 MPa) where little shielding effect was observed (Ln ∼ 13 mm).

The role of different modifiers on radiation shielding, optical, and physical properties for strontium boro-tellurite glass

In this work, we fabricated a new composition of TeO2–SrO–B2O3-MO (when RO = PbO, BaO, ZnO, and MoO3) by using the melt quench technique. The role of various modifiers on strontium boro-tellurite glass was explored by studying structural, optical, and gamma-ray shielding properties. The structure for the glass was examined by X-ray diffraction (XRD), Fourier Transform Infrared (FTIR), and physical properties. The band gap, Urbach energy, refractive index, cut-off wavelength, etc., were determined based on the absorption ultraviolet (UV) spectrum. The gamma-ray shielding properties were defined experimentally and compared with XCOM data. The XRD data illustrated the amorphous nature of all glass samples and the FTIR results affirmed the functional groups for borate and tellurite. The highest density was recorded for a sample containing lead (SBT:Pb), followed by barium (SBT:Ba), Molybdenum (SBT:Mo), and Zinc (SBT:Zn). The experimental mass attenuation coefficient (MAC)exp showed excellent compatibility with XCOM data, and the highest relative difference (RD) was 7.47. In conclusion, the SBT:Pb sample showed the maximum radiation shielding properties, making it a strong option for use in radiation shielding.

CD26/DPP-4 in Chronic Myeloid Leukemia.

Simple SummaryCD26/dipeptidylpeptidase IV (DPP-4) is a membrane-bound multifunctional protein expressed in many primary solid tumors and in hematological diseases. Recent investigations demonstrated its specific expression on leukemic stem cells (LSCs) of Chronic Myeloid Leukemia (CML) bone marrow (BM) and peripheral blood (PB) samples. Thanks to this evidence, CD26 has been considered a novel exclusive stem cell marker of CML. The aim of this review is to describe and analyze the role of CD26 in the context of hematological malignancies and specifically of CML and to highlight its potential clinical application for the management of this disease.CD26 expression is altered in many solid tumors and hematological malignancies. Recently, it has been demonstrated that it is a specific marker expressed on LSCs of CML, both in BM and PB samples, and absent on CD34+/CD38− stem cells in normal subjects or on LSCs of other myeloid neoplasms. CD26+ LSCs have been detected by flow-cytometry assays in all PB samples of Chronic-Phase CML patients evaluated at diagnosis. Additionally, it has been demonstrated that most CML patients undergoing Tyrosine Kinase Inhibitors (TKIs) treatment still harbored circulating measurable residual CD26+ LSCs, even when displaying a consistent deep molecular response without any significant association among the amounts of BCR-ABL transcript and CD26+ LSCs. Preliminary data of our Italian prospective multicenter study showed that CML patients with a poorer response presented with a higher number of CD26+ LSCs at diagnosis. These data confirmed that CD26 is a specific marker of CML and suggest that it could be considered for the monitoring of therapeutic responses.

Single Cell Transcriptome Profile of Myeloma and Immune Cell Characteristics in Patients with Durable Response Post CART

Background: Chimeric antigen receptor T cell therapy (CAR-T) is a recent therapeutic advance for the treatment of myeloma. Among the FDA approved and investigational CAR-T, primary refractory disease post CAR-T infusion is uncommon, however most patients eventually relapse. To gain insight to the myeloma cell and immune cell characteristics associated with early relapse (PD) versus durable response (DR), we examined myeloma cells and immune cells in both the bone marrow and blood by single cell RNA-seq.Method: Bone marrow aspirate and blood samples were collected prior to BCMA targeted CAR-T therapy. CD138 + and CD138 neg cells from bone marrow (BM) and peripheral blood mononuclear cells (PBMNC) were collected for 5' based scRNA-seq (10X Genomics). CD138 + cells were clustered by Harmony integration. Top expressing genes in unique clusters for PD and DR were analyzed by Ingenuity Pathway Analyses. To profile CD138 neg BM cells and PBMNC from myeloma patients, the scRNA-seq dataset was mapped to the human PBMC reference using the Seurat v4 reference-guiding approach. Differential gene expression between responders and non-responders was performed using the FindMarkers function for each of the immune cell clusters in the BM and PB.Results: BM and PB samples from 15 patients were analyzed. Among these, 5 patients had relapsed disease more than 1-year post CAR-T infusion (Durable Response, DR), and 10 patients had relapse within 1 year (early relapse, PD). Among these, CD138+ cells were available from 3 DR and 4 PD for analysis. Multimodal expression of BCMA were seen among the CD138+ cells across all samples. Using the mode of the highest expression level as a cut-off, patients with DR had a trend toward having higher fraction of BCMA high CD138+ cells compared to pts with PD. Clustering of all CD138+ cells by Harmony integration identified 12 distinct clusters, among which two clusters were unique to pts in DR, and one was unique to patients in PD. Ingenuity pathway analysis identified the top marker genes in these 3 clusters to be enriched in pathways for IL-15 signaling, BCR signaling, and primary immunodeficiency signaling. (Figure 1A-E.)Differential gene expression for immune cell clusters from CD138 neg BM cells and PBMNC were compared between pts in PD and DR. Interestingly, different patterns were seen in immune cells in PB and BM (Figure 1G). For example, compared to pts in PD, those with DR had decreased signaling in CD16 PB monocytes for interferon signaling and differentiation to macrophages, whereas the CD16 monocytes in the BM in pts in DR had increased expression of mitochondrial genes for anti-oxidant stress, polarization to M1 macrophage and anti-apoptosis. Similarly, pts in DR had CD8 Tcm in the BM with increased expression of genes involved in cell adhesion and anti-apoptosis, and CD4 Tcm in the BM with increased expression of genes in protein metabolism.Conclusion: scRNAseq analysis of myeloma cells suggest different tumor transcriptome profile may be identified in myeloma cells that could relapse early after CAR-T therapy. In addition, host immune profile both in the BM microenvironment and in systemic PB circulation could be associated with durable clinical response. [Display omitted] DisclosuresKapoor: Pharmacyclics: Consultancy; Glaxo SmithKline: Research Funding; Amgen: Research Funding; Sanofi: Research Funding; AbbVie: Research Funding; Ichnos Sciences: Research Funding; Sanofi: Consultancy; Regeneron Pharmaceuticals: Research Funding; Takeda: Research Funding; Karyopharm: Research Funding; Karyopharm: Consultancy; Cellectar: Consultancy; BeiGene: Consultancy. Dingli: GSK: Consultancy; Janssen: Consultancy; Sanofi: Consultancy; Novartis: Research Funding; Apellis: Consultancy; Alexion: Consultancy. Kumar: Bluebird Bio: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Novartis: Research Funding; BMS: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche-Genentech: Consultancy, Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Antengene: Consultancy, Honoraria; Carsgen: Research Funding; Oncopeptides: Consultancy; Beigene: Consultancy; Amgen: Consultancy, Research Funding; Merck: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Lin: Merck: Research Funding; Kite, a Gilead Company: Consultancy, Research Funding; Takeda: Research Funding; Bluebird Bio: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Sorrento: Consultancy; Juno: Consultancy; Legend: Consultancy; Gamida Cell: Consultancy; Vineti: Consultancy; Novartis: Consultancy; Janssen: Consultancy, Research Funding.

Follow-up Next Generation Sequencing (NGS) Analyses on Clonal Chronic Idiopathic Neutropenia (CIN) Patients: Insights in the Natural History of the Disease

▪Background: We have previously performed NGS analysis of genes that are recurrently mutated in myeloid malignancies in a cohort of patients with the diagnosis of chronic idiopathic neutropenia (CIN) according to previously reported criteria that largely overlap with those proposed for idiopathic cytopenia/neutropenia of undetermined significance (ICUS-N). We have thus estimated for the first time the frequency of clonal hematopoiesis in patients with CIN/ICUS-N (11.54%) and found that clonal CIN patients have a significantly higher risk of developing a myeloid neoplasm than those with no evidence of clonality (non-clonal). 1 However more longitudinal follow-up NGS studies are required for the tracking of clonal evolution and delineation of clonal CIN natural history.Aims: To conduct longitudinal follow-up NGS analyses in order to assess clonal evolution and associate the clinical significance of detected clonal aberrations with the risk of transforming to myeloid malignancy in clonal CIN clinical outcome.Methods: Genomic DNA was extracted from patients' BM or PB samples, sequencing libraries were prepared and subjected to targeted next generation sequencing (NGS) on an Ion S5 Prime Sequencer (Thermo Fisher Scientific) using a panel of 38 genes recurrently mutated in myeloid malignancies.Results: Follow-up analysis by NGS was performed in 16 clonal CIN patients (Figure 1). (Out of these 16 patients, follow-up NGS data has already been published in 9 patients, however additional timepoints were tested in 3 of them). 1 The median time between the first and subsequent analysis was 28.5 months (range 8-164 months). Ten of these patients carried the initial somatic mutations with only subtle changes in the size of clone as estimated by the variant allele frequency (VAF); the patients displayed absence of additional mutations and did not develop myeloid malignancy (Figure 1A-C, E-G, I, K, L, P). Two patients acquired a second mutation at follow-up. One of them still displayed stable disease course (Figure 1D) whereas the second eventually progressed to CMML (Figure 1H). The analysis also revealed that one patient lost the initial detected mutation at follow-up after 98 months (Figure 1J). Two patients who progressed to MDS/MPN and AML respectively, displayed a notable clonal expansion with additional mutations at the time of progression (Figure 1M and Figure 1N, respectively). Specifically, the patient who progressed to MDS/MPN acquired a mutation in JAK2 and ASXL1 while the patient who progressed to AML acquired the typical NPM1 p.L287fs mutation. The patient who developed MDS with multilineage dysplasia, carrying three mutations in DNMT3A and IDH1, showed a moderate increase in the VAF of these mutations at first follow-up (Figure 1O). The patient progressed to acute lymphoblastic leukemia (2 nd follow-up) with acquisition of additional truncating mutation in ETV6. Following treatment (3 rd and 4 th follow-up) mutation in ETV6 was lost, however the three mutations in DNMT3A and IDH1 persisted and their clone size increased.Conclusions: In the majority of patients tested for clonal evolution over time, most mutant clones appeared to be remarkably stable, with minimal VAF change, no acquisition of new molecular alterations and no progression to overt myeloid malignancy. Two CIN patients who transformed to a myeloid malignancy displayed a clonal expansion as was reflected by the increase of VAF and the development of additional mutations whereas in the third patient only a modest VAF increase was identified before malignant transformation. Finally in the patient bearing 4 mutations no progression to overt malignancy was observed after 12 months of follow-up. This ongoing study of sequential NGS analysis of CIN patients is anticipated to contribute to the better understanding and enrich further the knowledge on the natural history of this rare disease.

Combination of Melphalan Flufenamide and Anti-PD-L1 or Anti-CD38 Antibodies Enhances Anti-Myeloma Immunity

Introduction Melphalan flufenamide (Melflufen; Oncopeptides AB) is a novel enzyme-activated analogue of melphalan that enables a more rapid and higher intracellular accumulation of melphalan in tumor cells than is achievable by direct exposure to equimolar doses of melphalan. Our preclinical study showed that melflufen is a more potent anti-myeloma (MM) agent than melphalan, overcomes drug-resistance, and induces synergistic anti-MM activity in combination with bortezomib, lenalidomide, or dexamethasone (Chauhan et al, Clinical Cancer Res 2013;19:3019). However, the effect of melflufen on the immunosuppressive and tumor-promoting MM-host bone marrow (BM) accessory cells such as immunologically dysfunctional plasmacytoid dendritic cells (pDCs; CD123/IL-3Rα) remains unclear. Here, we utilized our coculture models of pDCs, T-, and NK cells with autologous patient MM cells to examine whether a combination of melflufen and immune checkpoint inhibitor anti-PD-L1 Ab, or daratumumab (anti-CD38 Ab), restores anti-MM immunity.Methods MM patient BM and PB samples (N=10; obtained after informed consent), and cell lines were used for the study. Minimally cytotoxic concentration of melflufen (0.1 µM) was used to assess immune functions. CTL/NK activity assays MM CD8 + T- or NK-cells were cultured with autologous pDCs (1:10 pDC:T/NK ratio) with melflufen (0.1 μM) alone, and with anti-PD-L1 (5 μg/ml) or anti-CD38 (0.5 μg/ml) Abs for 3-5 days; cells were washed to remove the drugs, and then cultured for another 24h with pre-stained target MM cells (10:1 E/T ratio; T/NK:MM), followed by quantification of viable MM cells by flow.Results 1) Both MM tumor cells and pDCs showed higher PD-L1 and CD38 levels vs normal plasma cells; 2) Treatment of MM patient total BM mononuclear cells or purified MM cells with melflufen (0.1 µM) increased PD-L1 expression on MM cells (1.84-fold, treated vs untreated; p<0.05). Importantly, treatment of MM cells with melflufen and anti-PD-L1 Abs enhanced anti-MM cytotoxicity; 3) Combination of melflufen and anti-PD-L1 Ab triggers activation of CD3 + T cells, evidenced by an increase in CD69 expression on CD3 + T cells (1.15-fold, treated vs untreated, p<0.05); 4) Combination of melflufen and anti-PD-L1 Ab induced a more robust autologous MM-specific CD8 + cytotoxic T lymphocyte (CTL) activity than melflufen alone (% MM lysis: melflufen: 20%; melflufen plus anti-PD-L1 Ab: 60%; n=5; p=0.013); 5) Meflufen and anti-PD-L1 also triggered pDC-induced NK cell-mediated MM-specific cytolytic activity (p<0.05); and finally, 6) Low doses of melflufen and anti-CD38 Abs enhanced pDC-induced NK cell-mediated MM-specific cytolytic activity (%Viability: melflufen: 75%; melflufen + anti-CD38 Ab: 12.5%; n=4; p=0.001).Conclusions The combination of melflufen and anti-PD-L1 increases pDC-induced T- and NK cell-mediated cytolytic activities against MM. Moreover, combined melflufen and anti-CD38 Abs modestly enhance pDC-induced NK cell-mediated MM-specific cytolytic activity. Our preclinical data suggest targeting PD-L1 in combination with melflufen as well as support an ongoing clinical trial of melflufen with anti-CD38 Abs to enhance anti-MM immunity. DisclosuresNupponen: Oncopeptides AB: Consultancy. Lehmann: Oncopeptides AB: Current Employment. Lindberg: Oncopeptides: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Other: Travel, Accommodations, Expenses; Camurus: Membership on an entity's Board of Directors or advisory committees, Other: Travel, Accommodations, Expenses; Affibody: Membership on an entity's Board of Directors or advisory committees. Gullbo: Oncopeptides AB: Consultancy. Richardson: Takeda: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; Janssen: Consultancy; Sanofi: Consultancy; Protocol Intelligence: Consultancy; Karyopharm: Consultancy, Research Funding; GlaxoSmithKline: Consultancy; Regeneron: Consultancy; AstraZeneca: Consultancy; Secura Bio: Consultancy; AbbVie: Consultancy; Oncopeptides: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding. Chauhan: C4 Therapeutics: Current equity holder in publicly-traded company; Oncopeptides: Consultancy; Stemline Therapeutics: Consultancy. Anderson: Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi-Aventis: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millenium-Takeda: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Scientific Founder of Oncopep and C4 Therapeutics: Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Membership on an entity's Board of Directors or advisory committees.

End of Treatment Peripheral Blood TCR Evaluation for Minimal Residual Disease Evaluation in Peripheral T-Cell Lymphomas

End of Treatment Peripheral Blood TCR Evaluation for Minimal Residual Disease Evaluation in Peripheral T-Cell Lymphomas

Prognostic Value of Minimal Residual Disease (MRD) Among Patients with Classical Hodgkin Lymphoma Undergoing Autologous Stem Cell Transplantation

Prognostic Value of Minimal Residual Disease (MRD) Among Patients with Classical Hodgkin Lymphoma Undergoing Autologous Stem Cell Transplantation

Stable Lead Isotopic Ratios as Indicator of Urban Geochemical Processes

The study is aimed to apply the Pb isotope fingerprinting technique for tracing pollution of urban surface deposited sediment (USDS). USDS reflect changes in the geochemical conditions occurring in the environment. USDS samples were collected in residential areas with multistory buildings in Russian cities: Magnitogorsk, Nizhny Tagil, Tyumen, Ufa, and Chelyabinsk. Elements concentrations and stable Pb isotopic ratios were measured in the samples. The reconstruction of the initial geochemical baseline (IGB) relationship between potentially harmful element (PHE) Pb and conservative lithogenic element (CE) Fe was carried out for USDS sample populations in the cities. The IGB reconstruction divided USDS sample populations into the groups of ‘polluted’ and ‘unpolluted’ with Pb samples. Analysis of elements concentrations and Pb isotope ratios in the groups of USDS samples showed different trends in altering geochemical conditions for metals in the surveyed cities. The USDS is characterized by a decrease in the isotope ratios of 206Pb/204Pb and 208Pb/204Pb as a result of soil pollution by vehicles during the period of using leaded gasoline.

The content and health risk assessment of selected elements in bee pollen and propolis from Turkey

This study aimed to determine the concentrations of potentially toxic elements (PTEs), including Al, As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Sn, V, and Zn, in pollen and propolis samples collected from migratory beekeeping areas in Turkey. A health risk assessment was performed to identify the potential risk of these PTEs to consumers in terms of public health. Concentrations of the elements were analyzed by inductively coupled plasma optical emission spectroscopy. The most abundant element in the bee pollen and propolis samples was Fe, with average concentrations of 47.3 and 390 mg kg−1, respectively, followed by Al, Zn, Mn and Sn. Concentrations of all elements except Cu, Mn and Sn were higher in propolis than in pollen. The concentrations of Fe, Al, and Zn in the propolis samples were over eight, seven, and two times higher than in the bee pollen samples, respectively. Se, Cd, and Hg in pollen and Se and Cd in propolis were below the detection limits. Pb, Co, and Cr were detected below 0.62, 0.06, and 0.91 mg kg−1, in pollen samples, respectively. Co and Hg were detected below 0.68, and 0.18 mg kg−1, in propolis samples, respectively. Detection of the PTEs contamination level and assessment of the health risks in pollen and propolis is necessary to ascertain quality and safety before consumption. In this study, we concluded that bee pollen and propolis may be good indicators for the screening of environmental pollution with PTEs and standards regulating acceptable concentrations of these pollutants in bee products should be established. In addition, it has been determined that consumption at the specified rates does not pose a risk. This study suggests the determination of admissible concentrations of PTEs in bee pollen and propolis.

Ofatumumab Plus Chemotherapy Followed By G-CSF For Mobilization Of Peripheral Blood Stem Cells (PBSC) In Patients With B-Cell Non-Hodgkin's Lymphomas (NHL)

![Graphic][1] Background The in-vivo use of anti-CD20 mAbs during PBSC mobilization has the potential to improve the cure rates following autologous stem cell transplantation in patients with NHL (Khouri et al. J Clin Oncol 2005). Ofatumumab, a type I human antibody, binds to a different epitope of CD20 than rituximab. We studied the feasibility of PBSC mobilization following ofatumumab with ifosfamide/etoposide (a standard chemo-regimen at our center). In addition, as the very late antigens (VLA)-4 and VLA-6 are expressed on hematopoietic stem cells (HSC) and both play a role in homing and engraftment of HSC, we studied these markers on HSC progenitor (CD34+) cells. Patients and Methods Eligibility criteria included patients with chemosensitive NHL with 100,000/mm3 and an ANC > 1500/mm3. Ofatumumab was given at 1000 mg on day 1 and 2000 mg of day 8. Chemotherapy (days 2-4) and G-CSF were used as per standard protocols at our center. Each patient provided a PB sample prior to mobilization and an aliquot of leukapheresis product (LP) after mobilization for detection of leukocyte subsets including T-, B-, natural killer (NK)- and dendritic cell (DC)-subsets, and VLA-4 and VLA-6 molecules on CD34+ PBSC by multicolor FACS analyses. Differences between median values of leukocyte subsets between LP and PB were determined using the Wilcoxon Signed Rank test. Results Seventeen patients were studied; Median age was 52 years (range, 25-68). Histologies included: Diffuse large cell (n=9), Follicular (n=4), Mantle cell (n=3) and double-hit lymphoma (n=1). Median number of prior chemotherapies received was 2 (range, 1-4). Fourteen (82.3%) patients mobilized PBSC after a median of 3 (range, 1-6) apheresis. WBC count at day one of collection was 19.6 (range, 4.8-31) k/μL. Three (17.6%) patients failed collection of at least 2x106 CD34+cells/kg but then mobilized successfully after receiving Plerixafor. All patients were able to receive an autologous transplantation with BEAM. Median number of CD34 infused was 5.56 x10^6/kg(range, 3.79-8.85). Median times to recovery of ANC > 500/mm3 and a platelet count of >20,000/mm3 were 11 days (range, 10-12) and 10 days (range, 9-15), respectively, following transplantation. With a median follow-up time of 9 months (range, 2-11 months) post-transplant, 16 (94%) patients remain alive in remission and 1 patient died of progression. Three patients experienced grade 2 toxicity with ofatumumab (hypertension, rash, and bone pain). None had > grade 2. Correlative studies were undertaken in 14 patients. LP had a significantly higher median WBC than paired PB samples (31.1 vs. 4.3 k/uL; p=0.001) but a significantly lower median percentage of lymphocytes (3.0% vs. 18.5%; p=0.001). Among the lymphocytes, LP had significantly higher percentages of CD3+ CD4+ T cells (56.9% vs. 46.7%; p=0.048) and CD3-CD16-CD56+ NK cells (13.7% vs. 7.24%; p=0.022), respectively than paired PB samples. There were no significant differences in total T (CD3+), T suppressor/cytotoxic (CD3+ CD8+) B (CD19+), and CD16+ NK cells. In addition, total dendritic cells (DC: Lin-HLA-DR+), and DC subsets comprised of myeloid-derived DC (mDC: CD11c+CD123-) and plasmacytoid-derived DC (pDC: CD11c-CD123+) were assessed in LP and PB samples. Compared with PB, LP had a significantly higher percentage of total DC (0.40 vs. 0.32; p=0.022) but significantly lower percentages of mDC (17.25 vs. 61.9; p=0.001) and pDC (13.35 vs. 21.85; p=0.035), respectively. The median percentage of HSC progenitors (CD34+ CD38dim) in PB was significantly lower in PB than in LP (12.3% vs. 46.4%, p=0.001). Moreover, LP specimens were assesssed for VLA-4/CD49d and VLA-6/CD49f expression. Median percentage of VLA-4 expression was significantly higher in LP than in PB samples (99.05% vs. 55.65%, p= 0.001). In contrast, the median percentage of VLA-6 expression was significantly lower in LP than in PB (22.15% vs. 60.3%, p=0.003). Conclusions PBSC mobilization with ofatumumab is feasible with minimal toxicity. This resulted in an increase in the recruitment of T-helper, NK, and DC subsets in the leukopheresis product, compared with levels in PB. Although others have reported a positive association between the expression of VLA-4 on CD34+ CD38dim cells and time to engraftment, we were unable to confirm this in our study. Since this is most likely due to the small sample size, additional studies are warranted. Disclosures: Khouri: GlaxoSmithKline: Research Funding. Off Label Use: Ofatumumab in stem cell mobilization. [1]: /embed/inline-graphic-2.gif

Simultaneous Analysis of Telomere Lengthes and Cell Surface Antigen in Leukemia by Multicolor Flow-FISH

During the last decade telomere length (TL) and telomerase have been discovered as new biological markers and potential new targets of cancer including malignant hematopoietic disorders. A FLOW-FISH method, i.e. quantitative fluorescence in situ hybridization (Q-FISH) in combination with flow cytometry (FCM), has been developed recently. Most overseas studies detect TL by FLOW-FISH without combined analysis of cell immunophenotype. We analyzed TL and cell immunophenotype simultaneously in the same sample by FLOW-FISH in order to combine the two markers to found one generally applied method for monitoring disease conditions or predicting clinical outcome in leukemia.Methods:HL60, Raji, Molt4 cells were cultivated. Each step and the conditions of the FLOW-FISH procedure were optimized, standardized and validated. The relative TL (RTL) of the leukemic sample cells compared to the control cells(Molt4).BM samples were derived from 34 leukemia patients. 20 normal PB samples from healthy individuals were set as control group.The BM or PB MNC were collected and used for FLOW-FISH. Meanwhile, the RQ-PCR measurements in the same samples were performed to test the correlation of the two methods.Results:Using various cell lines, multicolor FLOW-FISH was also successfully established.The median of RTL in 24 AL patients was 0.240, 2 CLL was 0.235, 3 CML-BP was 0.249, 5 CML-CP was 0.435, while that in 20 normal controls was 0.408.(P=0.001). The TL was shorter in patients with AL, CLL, CML-BP than in normal controls, otherwise, the CML-CP cases had no difference with the controls. 14 AL patients had the significantly longer telomere with clinical remissions than at impetus stage (P=0.01), and no significant difference was found between the 12 cases with clinical remissions and the normal controls(P=0.363). A high degree of correlation between the RTL obtained from FLOW-FISH and TL estimation by RQ-PCR existed (P=0.000).There was a significant negative correlation between TL and age in 20 normal controls(P=0.001), and the female had longer telomere than the male(P=0.037). However no statistic difference between TL and age or gender existed in 34 de novo leukemia patients. The TL of 24 AL cases had a negative correlation with peripheral white cells counts(P=0.002). Within 23 follow-up AL patients, the CR rate in cases with TL above the median was 81.82%, and 58.23% in the rest. Among 5 CML-CP patients who received imatinib treatment, 2 cases with the shorter telomere occurred transformation to BP in less than 6 months, the other 3 cases obtained CHR within 3 months, CCyR at 6 months and no transformation to BP was observed in our follow-up of 10 months.14 AL patients who undergone simultaneous analysis of TL and immunophenotype show the coincidence with the convention immunology in cell differentiation antigen expression. TL combined with cell differentiation antigen of 5 cases were dynamically detected at different courses of disease. It showed that TL of 2 cases with continued CR restored after CR and remained at normal level during remission, and no specific antigen expressed highly. However, telomere of 3 relapsed cases were significantly shortened at impetus stage, prolonged after CR and shortened again after relapse. In 2 relapsed cases, the telomere of related antigen positive cells shortened before the whole cells, and ahead of cell morphology change.Conclusions:The TL was significantly shorter in 34 de novo leukemia patients than in normal controls except the CML-CP cases. Restored telomeres were found with clinical remissions in 14 AL cases. The two methods of FLOW-FISH and RQ-PCR generated the same results and showed notable correlated.There was a significant negative correlation between TL and age in normal controls, and the female had longer telomere than the male. However no statistic difference between TL and age or gender existed in de novo leukemia patients. The TL of 24 AL cases had a negative correlation with peripheral white cells counts. It seemed the shorter telomere mean the lower CR rates in 23 follow-up cases with AL and the shorter duration of CP before onset of BP in 5 cases with CML.Simultaneous analysis of TL and cell differentiation antigen by multicolor FLOW-FISH made for overall monitor for leukemia and might be a new generally applied method for monitoring MRD in leukemia.

Ofatumumab, Very Late Antigen 4 (VLA4), and Dendritic Cells in Patients Undergoing Autologous Peripheral Blood Stem Cell (PBSC) Mobilization and Transplantation (ASCT) for Relapsed b-Cell Non-Hodgkin's Lymphomas (NHL)

▪Background: The in-vivo use of anti-CD20 monoclonal antibodies during PBSC mobilization has the potential to decrease the relapse rates following ASCT in patients with b-cell NHL (Khouri et al. J Clin Oncol 2005). We studied the feasibility of PBSC mobilization with G-CSF following ofatumumab with ifosfamide/etoposide (a standard chemo-regimen at our center). In addition to mobilizing CD34+ PBSCs, G-CSF mobilizes naïve T cells (CD3+ VLA4 low) and myeloid dendritic cells (mDC), as well as activates and matures the latter to become efficient antigen presenting cells. Patients and Methods: Eligibility criteria included patients with chemosensitive NHL with <5% marrow involvement at study entry, a platelet count of > 100,000/mm3 and an ANC > 1500/mm3. Ofatumumab was given at 1000 mg on day 1 and 2000 mg on day 8. Chemotherapy (days 2-4) and G-CSF were used per standard protocols at our center. Each patient provided a PB sample prior to mobilization and an aliquot of leukapheresis product (LP) after mobilization for detection of leukocyte subsets including T-, B-, - and DC-subsets, and VLA-4 and VLA-6 molecules on CD34+ PBSC by multicolor FACS analyses. Differences in values of leukocyte subsets between LP and PB were determined using the Wilcoxon Signed Rank test. Results: Fifty patients were studied; median age was 55 years (range, 25-70). Histologies included: diffuse large cell (n=33; 66%), including 9 double-hit; mantle cell (n=12; 24%) and indolent lymphoma (n=5; 10%). Median number of prior chemotherapies received was 2 (range, 1-5). Forty (80%) patients were in either first complete remission (CR1, n=18; 36%) or >CR1 (n=22; 44%); 9 (18%) in partial remission (5 of 7 were PET+), and 1 (2%) had stable disease. Forty-six (92%) patients mobilized PBSC after a median of 1.5 (range, 1-6) apheresis. Median WBC count at day one of collection was 17.7 k/μL (range, 3.6-52.3). Four (8%) patients failed collection of at least 2x106 CD34+cells/kg but then mobilized successfully after receiving Plerixafor. All patients were able to receive an ASCT. Median number of CD34 infused was 6.3 x10^6/kg (range, 3.2-16.9). Median times to recovery of ANC > 500/mm3 and a platelet count of >20,000/mm3 were 10 days (range, 9-12) and 12 days (range, 0-25), respectively, following transplantation. With a median follow-up time of 14.4 months (range, 1.2-50) post-transplant, the median overall survival (OS) and progression-free survival (PFS) rates have not been reached. The OS and PFS at the median follow-up time were 85% and 75%, respectively. Correlative studies have shown significantly higher percentages of CD3+ cells (82.2% vs. 74 % p<0.0001), CD4+ T cells (45.3% vs 34.2%; p<0.0001), and total DC (1.78% vs 0.41%; p<0.0001), in LP than paired PB samples, respectively. Differences in OS and PFS were evaluated for disease characteristics (histology, disease status, PET, number of prior therapies) and apheresis products. Whereas a significantly better OS rate was observed in patients who had apheresis products that contained naïve T cells (CD3+ VLA4 low) [p= 0.017 at the optimal cutoff value (figure) and 0.047 at the median], a better PFS was associated with apheresis products containing a higher percentage of mDCs (p=0.049). We were unable to confirm earlier reports of a positive association between the expression of VLA-4 on CD34+ CD38dim cells and time to engraftment.Conclusions:PBSC mobilization with ofatumumab is feasible with minimal toxicity, and resulted in a leukopheresis product with an increase in the recruitment of CD3+, CD4+ T cells, and DC, compared with levels in PB. Whereas superior OS is associated with naïve CD3+ VLA4 low T cells, better PFS was associated with higher percentage of mDCs. Since these observations were obtained with a small sample size, additional studies are warranted to validate our findings [Display omitted] DisclosuresKhouri:Novartis: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy.

A Phase I/II Study of Reduced Dose FCR (FCR-lite) in Combination with Lenalidomide Followed By Lenalidomide Maintenance (FCR2) for Initial Therapy of CLL: Final Results and Long-Term Follow-up

A Phase I/II Study of Reduced Dose FCR (FCR-lite) in Combination with Lenalidomide Followed By Lenalidomide Maintenance (FCR2) for Initial Therapy of CLL: Final Results and Long-Term Follow-up

Clinical Relevance of Minimal Residual Disease Monitoring in NPM1 Mutated AML: A Study of the AML Study Group (AMLSG)

Clinical Relevance of Minimal Residual Disease Monitoring in NPM1 Mutated AML: A Study of the AML Study Group (AMLSG)

Immune Predictors of Response to Pembrolizumab Monotherapy As Consolidation in Multiple Myeloma Patients: Results of the GEM-Pembresid Clinical Trial

Immune Predictors of Response to Pembrolizumab Monotherapy As Consolidation in Multiple Myeloma Patients: Results of the GEM-Pembresid Clinical Trial

A Phase I/II Study of Selinexor (SEL) with Sorafenib in Patients (pts) with Relapsed and/or Refractory (R/R) FLT3 Mutated Acute Myeloid Leukemia (AML)

Background: Responses to FLT3-inhibitors are usually transient due to emergence of resistance through the acquisition of kinase domain point mutations and other non-mutational mechanisms. SEL is a potent first-in-class Selective Inhibitor of Nuclear Export/SINE™ that exerted marked cell killing of human and murine FLT3 -mutant AML cells, including those with ITD, D835Y, ITD+Y842C or ITD+F691L mutations by modulating the cdk inhibitor p27 and anti-apoptotic Mcl-1 (Zhang W et al, Blood 2015 126). The combination of SEL+sorafenib had synergistic pro-apoptotic effects in FLT3-mutated AML cells by suppressing phosphorylation levels of FLT3 and its downstream signaling mediators ERK/AKT, and by inducing myeloid differentiation in ITD and D835 mutated cell lines.Methods: We designed a phase I/II trial of SEL with sorafenib for FLT3-ITD and/or -D835 R/R AML pts. In phase I, the primary objectives were to determine the maximum tolerated dose (MTD), the recommended phase 2 dose (RP2D), and dose-limiting toxicity (DLT) of the combination. In phase II, primary objectives included the rate of complete remission (CR) + CR with incomplete count recovery (CRi) + partial response + >50% blast reduction within 3 months of therapy initiation. Secondary objectives were safety and overall survival (OS). SEL was given orally twice a week for 3-weeks on and 1-week off in 28-day cycles and sorafenib was given continuously at a dose of 400mg twice daily from cycle 1 day 1 of SEL. In phase I, SEL dose was de-escalated in “3+3” fashion starting at dose level 0 and going down if DLTs were exceeded (dose levels 0, -1, -2 = 80, 60, 40 mg, respectively). Once MTD was established, the phase II enrolled pts in 2 cohorts: prior FLT3-inhibitor failure (cohort 1) and FLT3-inhibitor naïve (cohort 2). PB or BM samples were collected at pre-dose (C1D1), 24 h (C1D2) and day 28 (C1D28) of cycle 1, and apoptosis induction was determined by measuring annexin V positivity with FACS.Results: Fourteen pts with a median age of 71 years (range, 24-81) were enrolled. All pts had baseline next generation sequencing for AML specific mutations (Figure 1A). The median number of prior therapies was 3 (range, 1-5) as follows: salvage (S) 1: n=1, S2: n=5, S3+: n=8. 11 (79%) pts had prior FLT3-inhibitors: 1 prior FLT3-inhibitor (n=9); 2 prior FLT3-inhibitors: n= (2). Three pts had prior SCT. Four pts were treated at dose level 0 (SEL 80mg) with DLTs in 2 (Grade (G) 3 sepsis, n=1; G3 mucositis, syncope, adrenal insufficiency, n=1). Three pts were subsequently treated at dose level -1 (SEL 60mg) with no DLTs and this was established as the MTD/RP2D. Seven pts were treated in expansion: 6 had prior FLT3-inhibitor therapy (cohort 1) and 1 had no prior FLT3-inhibitor (cohort 2).The median duration of treatment for all 14 pts was 28 days (range, 14-122) and therapy is ongoing in 1 pt. All pts are evaluable for response. CRp/CRi and >50% blast reduction as best responses were seen in 4/14 (29%) and 2/14 (14%) pts, respectively. All 6 responders had a prior FLT3 inhibitor yielding a response rate of 55% (6/11) pts with prior FLT3 inhibitor therapy. 3 pts with CRp/CRi (2 with FLT3-ITD and 1 with ITD+D835) achieved negative RT-PCR for FLT3. (Figure 1B). One pt in CRi was bridged to SCT and remains alive at 1 year. The most common G 3/4 adverse events irrespective of causality were bleeding (gastrointestinal, n=4; intracranial, n=1), febrile neutropenia (n=4), pneumonia (n=3) and syncope (n=2). The reasons for discontinuing study therapy were disease progression (n=9; 64%), toxicity (n=3; 21%) and SCT (n=1; 7%). The median OS for pts on study was 3.5 months (range, 0.9-15). Translational analysis showed that SEL+sorafenib induced apoptosis in 4 of 5 tested patients, especially in the BM samples (Figure 1C)Conclusions: The combination of SEL and sorafenib is safe with clinical activity and apoptosis induction in R/R FLT3+ AML. The benefit was especially encouraging in pts exposed to prior FLT3 inhibitors, with a response rate of 55% (6/11; including 2 CRi and 2 CRp). The RP2D is 60 mg twice weekly of SEL and the study is accruing (NCT01607892). [Display omitted] DisclosuresDaver:Kiromic: Research Funding; Bristol-Myers Squibb Company: Consultancy, Research Funding; Daiichi-Sankyo: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Otsuka America Pharmaceutical, Inc.: Consultancy; Incyte Corporation: Honoraria, Research Funding; Pfizer Inc.: Consultancy, Research Funding; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding. Jabbour:Bristol-Myers Squibb: Consultancy. DiNardo:Celgene: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding. Cortes:BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; ARIAD: Consultancy, Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding. Kantarjian:Novartis: Research Funding; Pfizer: Research Funding; ARIAD: Research Funding; Amgen: Research Funding; Bristol-Meyers Squibb: Research Funding; Delta-Fly Pharma: Research Funding.

The Orally Available Selective Axl Inhibitor BGB324 Induces Diversification of the Immune Repertoire and Specific Changes in Plasma Biomarker Profiles

Introduction: The RTK Axl represents a novel therapeutic target in AML promoting proliferation and therapy resistance. In addition, Axl suppresses immune responses by decreasing antigen presentation and inhibiting NK cells. BGB324 is a first-in-class, orally available, selective Axl kinase inhibitor which is being explored as a therapy for solid tumours and myeloid malignancies. Selective blockade of Axl by BGB324 in AML is currently being explored in the Phase 1/2 trial BGBC003 (NCT02488408). Until now, n=25 patients have been treated in a classical 3+3 dose escalation design and treatment was generally well-tolerated (n=22 with AML and n=3 with MDS). Steady-state levels of BGB324 were achieved between three and six days after initiation of treatment. One AML patient achieved a CRi of five months, a second achieved a PR and another achieved clearance of circulating blasts accompanied by peripheral blood count recovery > three months (ORR 10%). Four additional AML patients (25%) experienced disease stabilisation for more than four months. Two MDS patients experienced a PR.Here, we report the spectrum of plasma proteins modulated by BGB324 in patients with AML/MDS and their relationship to treatment outcome and compound exposure. Furthermore, we analysed the B- and T- cell repertoire in order to assess potential effects of BGB324 on the immune response in AML patients.Methods: We used the DiscoveryMap panel (Myriad RBM) to measure plasma protein biomarker levels in a selection of patients pre-dose and at C2D1 and performed a cross-comparison with samples from an ongoing NSCLC study with BGB324 (BGBC004, NCT02424617) to identify candidates specifically modulated in AML. The TCRß repertoire was quantified by Next Generation Sequencing of DNA isolated from peripheral blood MNCs using an Illumina MiSeq sequencer. TCRß gene containing the entire Vß, Dß and Jß segments were amplified with BIOMED2-TCRß-A and -B primer pools. The IGH repertoire was analysed using BIOMED2-FR1/-FR3 primer pools. Using genomic DNA as template, the amplicons were tagged with Illumina adapters and indices in two consecutive PCR reactions. Demultiplexing and FastQ formated data output was generated by the MiSeq reporter. Analysis of TCRß and IGH data was performed on a Microsoft Cloud using our in-house analysis pipeline Pippa, which relies on MiXCR analysis tools.Results: Treatment with BGB324 significantly modulated Gas6, soluble Axl (sAxl), Angpt2, CXCL1 (GROalpha), SCGB1A1 (uteroglobin, club cell 10kDa protein) and PLAU (uPAR) levels in patient plasma. Of these, Gas6 was modulated after treatment with BGB324 in AML but not in lung cancer patients while CXCL1 and SCGB1A1 were only influenced in lung cancer patients. sAxl levels correlated with BGB324 exposure indicating that the shed Axl receptor represents a novel biomarker of Axl kinase inhibition.Furthermore, we studied the effects of BGB324 on the T- and B- cell receptor repertoire in n=5 available matched PB samples. We could demonstrate diversification of the TCR repertoire in PB by higher abundance of small T-cell clones at C2D1 with BGB324 compared to pre-treatment levels in three out of five analysed patients.The analyses of the IGH repertoire in the PB indicates higher diversity at C2D1 in two out of five available patients.Thus, treatment with BGB324 can increase the diversity of the IGH and TCRb repertoire in the PB of AML patients.Conclusion: BGB324 is well tolerated in AML patients, exhibits anti-leukemic activity and induces exposure-related modulation of biomarker candidates. Furthermore, BGB324 can induce a diversification of the TCR repertoire in AML patients and might hold potential as an immune-activating drug. DisclosuresLoges:BerGenBio ASA: Research Funding. Ben-Batalla:BerGenBio ASA: Research Funding. Micklem:BerGenBio ASA: Employment, Equity Ownership, Patents & Royalties. Brown:BerGenBIo ASA: Employment. Lorens:BerGenBIo ASA: Employment, Equity Ownership, Patents & Royalties, Research Funding. Yule:BerGenBIo ASA: Employment. Fiedler:Amgen: Patents & Royalties; Amgen, ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen, Pfizer: Research Funding; Amgen, Gilead, GSO, Teva, Jazz Pharmaceuticals: Other: Support for meeting attendance. Cortes:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Teva: Research Funding.

Hidden role of intrinsic Sb-rich nano-precipitates for high-performance Bi2-xSbxTe3 thermoelectric alloys

Nanostructuring is a universal strategy to improve the figure of merit (zT) of thermoelectric (TE) materials by the substantial decrease of lattice thermal conductivity due to the intensive scattering of phonons at interfaces. However, nanostructured bismuth antimony telluride alloys with foreign nanosized phases usually suffer from the degradation of carrier mobility at incoherent interfaces. The results reported here describe an approach to generate intrinsic Sb-rich precipitates that have a synchronal effect on micro-grained Bi0.5Sb1.5Te3 alloy. The designed formation of nanosized Sb-rich precipitates by dissolving excess Pb into Bi0.5Sb1.5Te3 matrix intensifies the phonon scattering and modulates the carrier transport simultaneously. By employing density functional theory calculation, transmission electron microscope and atom probe tomography, the role of Pb substituting for Sb site on the evolution of Sb-rich nanophase is clarified and the compositions and crystal structures of the precipitated Sb-rich phases are analyzed, revealing various interface structures. The optimized Bi0.5Sb1.5Te3 + 0.22 wt.% Pb sample shows a maximum zT value of 1.32 at 400 K with an outstanding average zT value of 1.2 over 300 K to 500 K. This work identifies the hidden role of intrinsic Sb-rich precipitates in Bi0.5Sb1.5Te3 alloys and provides an effective way beyond nanostructuring to develop high-performance bismuth antimony telluride thermoelectric alloys.