bluuebunny/crossref_metadata_2025 huggingface dataset:
Survival and Efficacy of Decitabine in Myelodysplastic Syndromes (MDS), Analysis of the 5-Day IV Dosing Regimen.
Authors: Kantarjian Hagop , Garcia-Manero Guillermo , O’Brien Susan , Estrov Zeev , Ravandi Farhad , Cortes Jorge , Shan Jianqin , Davisson Jan , Issa Jean-Pierre
Published: October 2019

An optimized dosing regimen for decitabine, an epigenetic therapy approved for the treatment of MDS, has been described recently based on the results of a single center, Phase II three arm adaptive randomization study (Kantarjian et al, Cancer2007;109:265–273). The results of this trial have been described in the context of a pooled data set from all three treatment arms; only a limited set of data specific to the 5-day IV dosing arm has been presented. Here, we provide an in-depth update of important efficacy and toxicity endpoints specific to the 93 patients treated within this trial with the 5-day schedule. 39/93 patients were not categorized (N/C) by IPSS due to exclusion from IPSS (extensive prior therapy, secondary MDS). Distribution by IPSS in the 54 categorized patients was: Intermediate-1 (INT-1) 26%, Intermediate-2 (INT-2) 50%, and High risk (HR) 24%. The median age was 65 years (range 37–83). Abnormal chromosomes were observed in 62%, 47% had >10% bone marrow blasts; 96% had various degrees of cytopenias. Secondary MDS was documented in 28 patients (30%). The median duration of MDS was 2.5 months. Prior therapy for MDS was given to 52 pts (56%): growth factors only in 34; chemotherapy ± growth factors 18. Overall 36 pts (39%) pts achieved CR, and 75 (81%) had a response by IWG criteria. The response rates by IWG criteria with IPSS subsets were: INT-1 71% (9 CR, 1 marrow CR); INT-2 70% (9 CR, 6 hematologic improvement [HI], 4 marrow CR); HR 62% (4 CR, 3 HI, 1 marrow CR); and N/C 72% (14 CR, 1 PR, 6 HI, 7 marrow CR). The median number of cycles was 8+. The median time to response was 2.3 months. The median duration of CR was 14 months (range 3 to 16+ months). The median time to acute myeloid leukemia (AML) transformation or death was 15 months. The median time to AML (censoring for death without AML) was 30 months. The median survival was 20 months, with 1- and 2-year survival rates of 61% and 41%, respectively. The side effect profile for the 5-day IV regimen was consistent with what has been previously reported, with Grade 3–4 drug-related extramedullary side effects being uncommon. The encouraging survival and response rates of the 5-day dosing regimen of decitabine in this poor-prognosis population, coupled with its manageable side effect profile, provide a beneficial therapeutic option and a foundation for further advances for patients with MDS.
Thrombin Generation in Severe Hemophiliacs with Different Clinical Phenotype.
Authors: Santagostino Elena , Mancuso Maria E. , Tripodi Armando , Chantarangkul Veena , Clerici Mariagrazia , Siboni Simona M. , Mannucci Pier M.
Published: October 2019

Introduction: Some severe hemophiliacs (FVIII/FIX<1%) exhibit a mild bleeding tendency, but the basis for this heterogeneous clinical expression is poorly understood. This study investigated the relationship between the values of endogenous thrombin potential (ETP) and clinical phenotype in severe hemophiliacs. The impact of FVIII/FIX gene mutations and thrombophilic polymorphisms into the modulation of the phenotype was also evaluated.

Methods: severe hemophiliacs older than 18 years without inhibitor history and treated on demand were eligible for inclusion in the study. Mild bleeders (MB) and severe bleeders (SB), representing the extremes of the clinical spectrum, were defined according to the following criteria: spontaneous bleeding episodes per year ≤2 (MB) or ≥25 (SB) and concentrate consumption <500 (MB) or >2000 (SB) IU/Kg/year. Patients who did not fit these criteria were considered as intermediate bleeders (IB). Plasma samples were obtained after a minimum wash-out period of 5 days. FVIII was measured by chromogenic assay. ETP was measured in platelet-rich plasma after addition of tissue factor.

Results: 22MB, 22SB and 28IB were enrolled. MB showed an older age at first bleed compared to SB (p < 0.005) and p for trend among the 3 groups was also significant (p < 0.05). The prevalence of severe FVIII/FIX gene defects (null mutations) was extremely low in MB (6%). ETP values were higher in MB compared with both IB and SB (p<0.05); p for trend among the 3 groups was also significant (p <0.05).

Conclusions: this study shows that the measurement of thrombin generation in platelet-rich plasma may allow to identify patients with mild bleeding diathesis among severe hemophiliacs, in contrast with the features of conventional functional assays.

SB (#22) IB (#28) MB (#22) P Age (yr) 38 (21–76) 38 (23–62) 32 (22–73) NS Age 1st bleed (yr) 1 (0–4) 2 (0–6) 3 (1–10) < 0.005 Bleeding episodes/yr 36 (25–60) 10 (3–20) 0 (0–2) < 0.0005 Factor use (IU/Kg/yr) 2207 (2040–8696) 1068 (207–2400) 60 (25–487) < 0.0005 Null mutations (%) 59 70 6 < 0.005 PTG20210A (%) 0 7 5 NS FV Leiden (%) 5 7 0 NS Median ETP (nM) 414 478 850 < 0.05
Who Gets Inhibitors? Predicting Inhibitory Antibodies in Children with Severe Hemophilia A.
Authors: ter Avest Petra C. , Fischer Kathelijn , Santagostino Elena , van den Berg Marijke H. , van der Bom Johanna G.
Published: October 2019

Background. Replacement therapy in severe hemophilia A patients is complicated by formation of inhibitors in around 25% of children. Identification of patients at the highest risk may help to tailor personalized treatment strategies.

Objectives. To develop a scoring system that may be used to identify patients at the highest risk of inhibitor development at first treatment.

Methods. We used the data from a retrospective multicentre cohort study (the Canal cohort) of patients with severe hemophilia A (factor VIII (FVIII)less than 0.01 IU/ml), born 1990–2000, followed at least until their 50th exposure day. Presence of inhibitor was defined as twice a positive inhibitor titer and a decreased FVIII recovery. Based on the coefficients of a logistic regression model, a weighted risk-score was developed. Shrinkage of regression coefficients was used to control for overfitting. The discriminative ability of the risk-score was expressed as the area under the curve (AUC) of a receiver operating characteristic curve.

Results. Out of the 366 patients from the Canal cohort, 284 children were selected of whom 78 developed an inhibitor (27%). Logistic regression analysis revealed 3 independent risk factors for inhibitor development: positive family history, intensive treatment (at least 5 consecutive days) at the first FVIII exposure, and high risk FVIII gene mutations. Table 1 presents odds ratios of the uni- and multivariate analyses, and the risk score. The AUC for the risk-score was 0,724. Table 2 shows that the model is able to separate high and low risk patients from patients with an intermediate risk of 25%.

Conclusions. The risk score includes positive family history of inhibitors, high risk factor VIII gene mutation and intensive treatment at first FVIII exposure. This risk score can recognize patients with a doubled risk for inhibitor development and may be used to guide inhibitor preventive treatment strategies.

Table 1. Uni- and Multivariate analysis and risk-score. OR (CI), Univariate OR (CI), Multivariate p-value Risk-Score CI = confidence interval 95% Positive Family History of Inhibitors 4.0 (1.7–9.4) 3.0 (1.2–7.7) ,022 3 High risk FVIII Gene Mutation 3.6 (1.8–7.2) 3.7 (1.8–7.9) ,001 4 Intensive Treatment at 1st FVIII exposure 6.8 (3.6–12.9) 7.2 (3.4–15.1) ,000 6 Table 2. Calibration of the risk-score. Model Total n° patients Predicted n° Inhibitors Observed n° Inhibitors Positive Predictive Value Negative Predictive Value LR= Low Risk, MR= Medium Risk, HR= High risk LR:0 72 7 6 0,34 0,92 MR:3–4 153 39 38 0,25 0,69 HR: >4 59 32 34 0,58 0,80
Effects of Recombinant Factor VIIa (NovoSeven®) on Restoring Thrombin Generation in Patients with Hemophilia A and Antibodies to Factor VIII.
Authors: Eichinger Sabine , Lubszcyk Barbara , Zwiauer Karl , Gleiss Andreas , Quehenberger Peter , Kyrle Paul Alexander
Published: October 2019

Introduction: Development of antibodies (ab) against factor VIII (FVIII) is a serious complication of replacement therapy in patients with hemophilia A. In case of bleeding patients with FVIII ab are treated with agents that induce hemostasis independently of FVIII. Recombinant activated factor VIIa (rVIIa) shows clinical efficacy, but its effects on hemostatic system need still to be fully elucidated.

Methods: In an open controlled study, we investigated thrombin generation (peak thrombin) and parameters of coagulation activation [D-Dimer, prothrombin fragment F1+2 (F1+2)] in 5 patients with hemophilia A and FVIII ab, and in 5 healthy age-matched controls before and after intravenous bolus infusion of rVIIa (90 μg/kg bodyweight NovoSeven®, NovoNordisk, Denmark) (in hemophiliacs only). All parameters were measured in plasma before and 0.5, 1, 2, 3, and 4 hours after rVIIa infusion by use of commercially available assays (Technothrombin®TGA, Technoclone, Austria; Asserachrom®D-Di, Diagnostica Stago, France; Enzygnost F1+2, Dade Behring, Germany).

Results: At baseline, hemophilia A patients had markedly lower mean (min-max) peak thrombin levels than controls [0.12 (0.0–0.6) nM vs. 186.9 (116.0–254.4) nM]. Mean (min-max) F1+2 levels did not significantly differ between patients and controls [160.7 (89.8–331.3) pmol/l vs. 160.8 (104.4–242.3) pmol/l]. Notably, D-Dimer levels were significantly higher in hemophiliacs than controls [1087.5 (174.8–3882.4) ng/ml vs. 146.3 (87.2–289.8) ng/mL]. FVIIa levels reached a mean (min-max) maximum of 28 (24–32) U/ml after 0.5 hours in all patients. After infusion, a considerable increase in mean (min-max) peak thrombin levels to 40.7 (28.3–51.6) nM was seen. Time to maximum levels was 30 minutes in three patients and 60 minutes in two. For each of the five patients the peak thrombin level was substracted from the level of its matched control at the same time point. The mean of these differences was 168.7 nM (95% CI 82.6–254.8), which translates into 80.2% (95% CI 65.4% – 88.6%) lower peak thrombin levels in haemophiliacs with FVIII ab. F1+2 significantly increased in all patients [mean (min-max) maximum levels 292.5 (175.1–464.3) pmol/l]; time to maximum levels varied from 2 to 4 hours. D-Dimer levels remained almost unchanged in all patients.

Conclusion: Patients with hemophilia A and FVIII ab have low in vitro thrombin generation and F1+2 levels. After rVIIa infusion, coagulation activation as measured by F1+2 levels is slightly increased, and thrombin generation capacity is restored by 20% compared to healthy controls. Measurement of peak thrombin could be useful to monitor procoagulant treatment of patients with hemophilia A and FVIII ab.
The Activity and Toxicity of Dasatinib in Relapsed or Plateau-Phase Multiple Myeloma.
Authors: Wildes Tanya M. , Ruddell Alisa , Gao Feng , Uy Geoffrey , Cashen Amanda , Abboud Camille , Westervelt Peter , Dipersio John F. , Vij Ravi
Published: October 2019

Dasatinib (Sprycel®) is an orally administered inhibitor of multiple tyrosine kinases, which has been approved for the treatment of CML in chronic phase, accelerated phase or blast phase with resistance or intolerance to prior therapy including imatinib. In vitro studies have demonstrated that dasatinib inhibits the viability of multiple myeloma cell lines. Based on the in vitro activity of dasatinib against multiple myeloma, we initiated an open label, single-arm Phase II trial of dasatinib in relapsed, refractory or plateau-phase multiple myeloma; planned enrollment is 21 patients (pts). Treatment consists of dasatinib 70 mg p.o. twice daily continuously. The primary endpoint of the study is the overall response rate (CR+PR). Secondary endpoints include the time to response, duration of response, time to progression as well as tolerability and safety of dasatinib in this population. To date, 13 pts have been enrolled. The median age is 58 (range 38–76). Stage at diagnosis was: IIA (1 pt), IIIA (11), IIIB (1); 9 pts had IgG paraprotein and 4 had IgA. Median beta2-microglobulin at study entry was 3.2 (range 1.2–13). The median number of prior therapies was 2 (range 1–14); 12 pts had undergone prior autologous stem cell transplant. At enrollment, 7 pts had progressive disease and 6 pts had plateau phase disease, defined as at least 3 months with a stable paraprotein level (±25%) following the most recent therapy. At a median follow-up of 39 days (range 1–134 days), results are as follows: 4 patients have stable disease (SD); 7 patients had progressive disease (PD); 1 pt discontinued therapy due to toxicity (Grade II diarrhea, flushing and fatigue); 1 pt is not yet evaluable for response. Of those who remain on therapy, the median time on therapy is 39 days (range 1–134); 4 of these 5 patients had plateau phase disease at enrollment. Of those who discontinued therapy for PD, median time on therapy was 46 days (range 15–91); 6 of these 8 patients had PD at the time of enrollment. Toxicities have been manageable. Hematologic toxicities were more pronounced in the patients who had cytopenias at baseline. There was 1 episode of Grade III neutropenia, 4 episodes of Grade III anemia, 2 episodes of Grade III thrombocytopenia, and 3 episodes of Grade IV thrombocytopenia. Grade III non-hematologic toxicities have included: pneumonia (3 pts), acute renal failure (2), headache (1), epistaxis (2), febrile neutropenia (1), pulmonary edema (1), GI bleed (1), fatigue (2), hypertension (1), and hip pain (1). There was 1 episode of grade IV hypoglycemia. Enrollment and evaluation of responses/toxicities are ongoing. The protocol is being amended to allow for dose escalation to dasatinib 100 mg b.i.d. for stable disease at 8 weeks on therapy.
In Vivo Expansion of Megakaryocyte Progenitors in CD26−/ − Mice: Implications for Novel Post-Transplant Therapeutics.
Authors: Kidd Shannon , Bueso-Ramos Carlos E. , Paganessi Laura A. , Fung Henry , Gregory Stephanie , Christopherson Kent W.
Published: October 2019

Hematopoietic stem cell transplantation (HSCT) is a successful treatment option for patients with malignant or non-malignant severe hematologic diseases. Readily available umbilical cord blood (CB) has emerged as an important donor source that has a lower histocompatability requirement and carries a reduced risk of graft vs. host disease. However, a frequent complication of CB use is delayed hematopoietic recovery, in particular long lasting severe thrombocytopenia (median of 117 days to platelet recovery, COBLT 2005). To overcome this clinically, effective strategies for enhancing megakaryopoiesis, thrombopoiesis, or both are needed. We have previously described the importance of CD26 (dipeptidylpeptidase IV) in engraftment (Christopherson, KW 2nd, et al, Science2004. 305:1000–3). However, the involvement of CD26 in megakaryopoiesis has not been investigated. We hypothesized that CD26 acts to suppress megakaryopoiesis and that removal of CD26 activity would result in expansion of the megakaryocyte progenitor population in vivo. To test this hypothesis, we evaluated megakaryocyte (MK) development in the context of development of other mature blood cells in CD26 deficient (CD26−/ −) mice as compared to control C57BL/6 mice. Histological analysis of formalin fixed paraffin embedded tissue sections and peripheral blood cytopsins revealed an increased presence of MK in the bone marrow, spleen, thymus and peripheral blood. However, complete blood counts (CBC) suggest no difference in white blood cell, neutrophil, lymphocyte, monocyte, eosinophil, basophil, and platelet counts. Flow cytometric analysis also revealed no significant changes in CD3, CD4, or CD8 T-cells; B220 B-cells; and Gr-1/Mac-1 granulocytes/neutrophils in the bone marrow, spleen, thymus, or peripheral blood as appropriate. There was also no change in the percentage of peripheral blood CD41 MK but there was a increase from 0.09% in C57BL/6 BM to 0.26% CD41+Sca-1+c-kit− MK progenitors in the CD26−/ − BM (P≤0.05). Methylcellulose based myeloid progenitor assays did also reveal respective CFU-GM, BFU-E, and CFU-GEMM per femur values (mean±SEM) of 24480±1426, 1448±154.86, and 852±41 for C57BL/6 BM cells; 24451±1342, 974.29±81, and 1634±177 for CD26−/ − BM cells. This represents a 92% increase in CFU-GEMM and corresponding 30% decrease in BFU-E in the CD26−/ − mouse BM (P≤0.01, n=7 mice/group). Collagen based megakaryocyte progenitor assays (CFU-MK) revealed a 25% increase from 4455.00±207.62 colonies/femur in C57BL/6 BM to 5555.00±608.12 in CD26−/ − BM (P≤0.05, n=3 mice/group). These results establish a basis on which to propose that CD26 may act to regulate early events in megakaryocyte progenitor formation and function. They also suggest that the use of CD26 inhibitors may have a beneficial effect on improved megakaryocyte progenitor function and/or reconstitution post-transplant.
Ex Vivo Treatment of Human Cord Blood with dmPGE2 Can Safely Increase the Potential for Hematopoietic Engraftment.
Authors: North Trista E. , Goessling Wolfram , Armant Myriam , Kao Grace S. , Silberstein Leslie E. , Ritz Jerome , Zon Leonard I.
Published: October 2019

Hematopoietic stem cells (HSCs) are commonly used in transplantation therapy to rescue the hematopoietic and immune systems following systemic chemotherapy or irradiation. However, some patients receive inadequate numbers of HSCs and this often results in delayed reconstitution of hematopoiesis and immune function and associated toxicities. We previously demonstrated that a stabilized derivative of prostaglandin (PG) E2 increases vertebrate HSCs both in vivo and in vitro. 16,16-dimethyl PGE2 (dmPGE2) significantly increased HSCs during zebrafish embryogenesis and in the adult marrow following injury. Incubation of murine embryonic stem cells with dmPGE2 during embryoid body differentiation resulted in a dose-dependent increase in hematopoietic colonies, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Finally, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in engrafting cells in a limiting dilution competitive repopulation assay. No negative effects on serial transplantability of HSCs were observed in these animal models. To investigate the therapeutic potential of PGE2 for the amplification of blood stem cells, we exposed human cord blood (hCB) cells to dmPGE2 in vitro and measured the effects on stem and progenitor populations both in vitro and in vivo. Red cell depleted umbilical cord blood specimens, cryopreserved for clinical use, were thawed and divided for parallel processing. Ex vivo treatment of hCB cells for 1 hour with dmPGE2 in dextran/albumin had no negative impact on absolute cell count or the viability and relative distribution of both CD45 and CD34 positive cells compared to vehicle treated control hCB cells. Significantly, hCB treated with dmPGE2 produced enhanced numbers of GM and GEMM colonies in methylcellose CFU-C assays compared to controls. Human CB cells treated ex vivo with dmPGE2 for 1 hour and transplanted at a dose of 20 million live CD45+ cells per recipient were capable of repopulating NOD/SCID mice after sublethal irradiation. In comparative studies at 6 weeks post transplantation, human CD34+ and CD45+ cells could be detected in the marrow (>2%) of dmPGE2 treated (4/8) and control treated (1/6) recipients. Long-term and competitive transplantation experiments to assess the effect of dmPGE2 treatment on functional HSCs are currently in progress. Our data suggests that treatment of human cord blood products with dmPGE2 will be both safe and effective in achieving expansion of hematopoietic stem cells for transplantation in the clinical setting. TE North and W Goessling contributed equally to this work.
Exchange of microRNAs between Breast Cancer Cells and Bone Marrow Stroma: Potential for Avenues To Revisit Hematopoietic Stem Cell Transplantation for Breast and Other Cancers That Show Preference for Bone Marrow.
Authors: Taborga Marcelo , Rameshwar Pranela
Published: October 2019

Previous failure of autologous bone marrow transplantation as a viable treatment of breast cancer opens the field for investigational studies since the bone marrow, as a source of cancer metastasis, remains a clinical dilemma. This study hypothesizes that a subset of breast cancer cells enter the bone marrow at an early period of disease development, perhaps prior to clinical detection, where they evade chemotherapeutic damage even at high doses. An understanding of this cell population might be crucial for the future success of bone marrow transplants in patients suffering from breast cancer metastasis to the bone marrow. In vivo and in vitro studies have demonstrated gap junctions between bone marrow stroma and breast cancer cells. The gap junctions can passage CFDA cell tracer between stroma and cancer cells. This exchange can be blocked by the gap junctional intercellular communication inhibitor, 1-Octanol, suggesting functional gap junctions. We have now implemented quantitative studies to determine the role of specific microRNAs (miRNAs) in blunting of mRNA translation of the Tac1 gene in cancer cells. The first set of studies optimized the effects of miRNAs, 130a and 206 in the suppression of Tac1 mRNA in the low invasive T47D and the aggressive MDA-MB-231 breast cancer cell lines. The miRNAs were transfected into cells containing the pMIR-R/Tac1/SG vector, which includes the 3′ UTR of Tac1 mRNA. Luciferase activity in cell lysates showed synergism between the two miRNAs, indicating that the predicted miRNAs can inhibit Tac1 translation. The next set of studies determined whether miRNAs 130a and 206 can cross gap junctions between stroma and cancer cells to suppress Tac1 mRNA. The miRNAs were transfected in one cell type and pMIR-R/Tac1/SG in the other cell type. Luciferase activity and parallel studies with 1-Octanol verified movement of miRNAs via the gap junction. These studies recapitulate the downregulation of endogenous Tac1 and other cytokines in the cancer cells. In summary, this study shows a role for miRNA exchange, between bone marrow stroma and breast cancer cells, as a mechanism by which the cancer cells might evade detection and current methods of treatment. An understanding of these mechanisms might lead to new methods to remove cancer from bone marrow in future therapies.
Large Regions of Uniparental Disomy (UPD) Establish Clonal Hematopoietic Stem Cell Selection in a Subset of Myelodysplastic Syndrome (MDS) Patients with Normal Bone Marrow Cell Karyotypes.
Authors: Heinrichs Stefan , Kulkarni Rima V. , Bueso-Ramos Carlos E. , Loh Mignon , Levine Ross , Li Cheng , Neuberg Donna S. , Kornblau Steve M. , Issa Jean-Pierre , Gilliland D. Gary , Estey Elihu H. , Look A. Thomas
Published: October 2019

Myelodysplastic syndromes (MDS) comprise a large group of hematological diseases sharing clinical features, but the diverse mechanisms underlying their molecular pathogenesis remain largely undefined. In this study, we used high-density 250K SNP arrays to analyze matched constitutional (buccal) and whole bone marrow DNAs from 36 patients with MDS. In 14 cases, we also selected CD34+ stem and progenitor cells for comparison. 23 of the cases were cytogenetically normal. Whereas regions of genomic amplification were rare, we found distinct regions of heterozygous deletion in 9 patients, most frequently targeting chromosomes 5q, 7q, 17p and 20q. In one patient with a large heterozygous 5q deletion, a smaller region in 5q34 was homozygously lost. Based on genotyping we could identify regions of uniparental disomy (UPD) in 4 patients, three of whom had normal bone marrow karyotypes. Two of the regions of UPD targeted chromosome 7q, suggesting that the frequency of 7q deletion is underestimated by conventional cytogenetics, and that a mutation has occurred in these cases within the reduplicated region involved in UPD. Comparison with constitutional DNA was critical for accurate SNP analysis in MDS, allowing us to focus on acquired clonal abnormalities and control for changes due to inherited copy number variation or unusual regions of germline homozygosity. For example, one case had several large blocks of germline homozygosity distributed over various chromosomes, and by comparison with the buccal DNA we were able to discern that these regions did not result from clonal somatic abnormalities associated with MDS. The analysis of CD34+ cellular DNA in 14 cases revealed that all aberrations identified in the stem and progenitor fraction were also identified in whole bone marrow DNA. This finding emphasizes the dominance and stem cell involvement of the abnormal clone in MDS, which makes purification of CD34 cells unnecessary for SNP array analysis in this disease. Most, but not all, clonal deletions evident by cytogenetic analysis were detected as copy number alterations by SNP array, possibly indicating that such abnormalities occurred in subpopulations of the cytogenetically analyzed MDS cells. Thus, SNP array and cytogenetic analysis are complementary methods for the identification of abnormal clones in MDS patients, each detecting overlapping but distinct subsets of patients. Our data indicate that SNP array analysis should be performed in all patients with MDS and normal bone marrow cytogenetics, in order to help discriminate patients with UPD indicating cell autonomous clonal stem cell malignancy from those who might have extrinsically mediated bone marrow failure syndromes.
Evaluation of an Automated Ficoll Procedure To Enrich for Mononuclear Cells for Use in Clinical Trials.
Authors: Sepulveda Carmen A. , Richman Sara J. , Kaur Indreshpal , Shen Anna , Khorana Sangeeta , O’Connor Sean L. , Champlin Richard E. , Shpall Elizabeth J. , McMannis John D.
Published: October 2019

Mononuclear cell fraction (MNC) enrichment from hematopoietic progenitor cells (HPC) cells is required for a number of clinical applications. Previously, we have used a manual method for enrichment of these cells. We evaluated an automated procedure for better consistency and patient safety (reduce risk of contamination). We have been using the Sepax (BioSafe) for the past 18 months to process Cord Blood (RBC depletion) before cryopreservation. Since the instrument can also do density gradient enrichment of mononuclear cells, we evaluated the ability to process different sources of hematopoietic progenitor cells (HPC) on the Sepax.

Methods: Bone marrow, non-mobilized apheresis and cord blood were evaluated as a source of HPC. Briefly, 100ml of Ficoll solution were added to the chamber of the Sepax disposable. After initiating centrifugation, 50–110 ml of HPC were then overlayed onto the density gradient. The cells were fractionated, mononuclear cell fraction isolated, and then washed three times before final resuspension in 50ml of Saline/5% albumin. In some experiments the HPC were divided into two fractions so that a direct comparison of the Manual vs Automated enrichment procedure could be compared. Finally, a recent publication from Seeger et al (European Heart Journal28:766, 2007) suggested different density gradient solutions have an effect on MNC enrichment. Therefore, four different brands of density gradient solution (GE Ficoll-Paque Premium, Sigma Histopaque 1077, Accurate Lymphoprep and Lonza Lymphocyte Separation media) were evaluated to determine if there was a differential separation of subsets of cells.

Results: The total nucleated cell (TNC), MNC and CD34 recoveries after MNC enrichment were similar for either bone marrow or cord blood HPC comparing the manual procedure and the automated procedure. The TNC and MNC recovery was higher for apheresis products (66% and 69% respectively) than the other two sources of HPC. This most likely reflects the enriched MNC starting population from an apheresis device. We evaluated the TNC and MNC recoveries over a range of initial TNC (7.5–20 × 108 cells) but could not see an effect of either cell number or cell concentration on recoveries. Nor could we see an effect of initial cell volume (50–100 mls) on recovery of cells post processing. Finally, we could not see a difference in either TNC or MNC recovery when the four different sources of density gradient solution were compared.

Conclusion: We have shown that the Sepax instrument can be used for enrichment of mononuclear cells from HPC, Marrow, HPC, Apheresis and HPC, Cord. The benefits of an automated system are

consistency, functionally closed system which reduces possibility of contamination documentation of run parameters and shorter processing time.
Genes Identified in a Hemopoietic Lineage Switch Influence Transcription.
Authors: Winteringham Louise N. , Endersby Raelene , Majewski Ian J. , Beaumont Jennifer , Kobelke Simon , Lalonde Jean-Philippe , Klinken Svend Peter
Published: October 2019

The J2E erythroblastoid cell line responds to erythropoietin (Epo) by morphological maturation and hemoglobin synthesis. However, on rare occasions, these cells have undergone a spontaneous lineage switch and display features of monoblastoid cells which do not respond to Epo. Amongst the genes up-regulated in the monoblastoid variants were Hemopoietic lineage switch (Hls) 5 and 7. Hls5 is a recently identified member of the RING finger, B Box, Coiled coil (RBCC) or tripartite motif (TRIM) family, which includes PML, a gene involved in acute promyelocytic leukaemia. Hls7 is the murine orthologue of Myeloid leukaemia factor 1 (Mlf1), a gene involved in a t(3;5), associated with acute myeloid leukaemia. We have shown previously that Hls7/Mlf1 imposes a dramatic phenotypic change upon the erythroid cells, rendering them monoblastoid (Williams, J. et al EMBO 1999). We have studied the role of Hls5 and Mlf1 in erythroid commitment and differentiation. Ectopic expression of Hls5 inhibits globin production in erythroid cells and suppresses development of B-FUE and C-FUE. A yeast-two-hybrid screen identified FOG-1 as a binding partner of Hls5. Significantly, FOG-1 is a transcriptional co-regulator of GATA-1, which controls globin gene expression. While Hls5 is able to enhance the repression of GATA-1 activity imposed by FOG-1, it is also able to repress GATA-1 transcriptional activity in the absence of FOG-1. Using electrophoretic mobility shift assay we have shown that Hls5 is able to reduce GATA-1 binding to DNA in a dose dependant manner. This observation that Hls5 reduces GATA-1 binding to promoter elements is mirrored by chromatin immunoprecipitation assays. Expression of MLF1 is highest in CD34+ cells, but is markedly down regulated during erythroid differentiation. Microarray analysis identified a number of known transcriptional regulators differentially expressed in the presence of Mlf1 including ets1, Myc intron binding protein and Tbr2. Mlf1 is able to bind DNA and luciferase reporter assays demonstrated that Mlf1 is able to affect transcription. In addition, Mlf1 interacts with a novel member of the hnRNP family viz Mlf1 associated nuclear protein (Manp). Manp binds to DNA, is able to influence the subcellular localisation of Mlf1 by translocating Mlf1 from the cytoplasm to the nucleus. Importantly, Manp also has an affect on transcription. These data demonstrate that both Hls5 and Mlf1 affect transcription of genes associated with erythroid differentiation.
Expression of Endothelial Protein C Receptor Confines Hematopoietic Stem Cell in Murine Fetal Liver.
Authors: Iwasaki Hiroko , Arai Fumio , Kubota Yoshiaki , Suda Toshio
Published: October 2019

Endothelial protein C receptor (EPCR or CD201) is expressed in the endothelial cells of large blood vessels, such as aorta, and contributes to the natural anticoagulant and anti-inflammatory system by activating protein C. Recent study showed that EPCR is also expressed in murine bone marrow, and that the majority of the EPCR-positive cells are the hematopoietic stem cells (HSC) in quiescent state, defined as the side population (sp) fraction in flow cytometry, suggesting the possibility of EPCR involvement in the hematopoiesis. In the murine mid-embryonic stage, on the other hand, the main location of hematopoiesis is fetal liver (FL), where the HSCs are highly proliferative. In this period of development, there are no quiescent HSCs, which is different from adult bone marrow. It is not clarified whether EPCR has any relevance to the fetal hematopoiesis. Here, we demonstrate that EPCR is involved in the hematopoiesis in FL. Immunostaining of murine FL (E12) clearly showed the EPCR-positive cells, and they were located adjacent or connected to the Lyve-1-positive sinusoidal endothelial cells. Some of those were endothelial-like, and others were in round shape, co-expressing c-Kit. In the HSC-enriched Lineage−Sca-1+c-Kit+ (LSK) fraction, the proportion of the EPCR-positive cells was approximately 30%. Sorted EPCR+/LSK and EPCR−/LSK cells were examined for the capability of bone marrow reconstitution and multilineage differentiation. In colony assay, the EPCR+/LSK cells exhibited higher proliferation and differentiation capability into the full range of the hematopoietic cells, including the megakaryocytes and erythroids, while most of the EPCR−/LSK-derived colonies were granulocytes and macrophages. Furthermore, the EPCR+/LSK and EPCR−/LSK cells isolated from donor FL were transplanted with the bone marrow-derived competitor cells into the lethally irradiated recipient mice, resulting in the striking difference between two fractions, i.e., robust long-term reconstitution of EPCR+/LSK cells and almost no reconstitution of EPCR−/LSK cells. The EPCR+/LSK cells were serially transplantable. These data collectively conclude that EPCR-expressing cells in LSK fraction of E12.5 FL exclusively exhibit the capability for long-term bone marrow reconstitution and self-renewal, as well as multilineage differentiation. Thus, the EPCR+/LSK fraction is the highly HSC-enriched population. The function of the endothelial molecule in HSCs will be discussed.
The Vent-Like Homeobox Gene VENTX2 Is Expressed in Human Hematopoietic Progenitor Cells and Promotes Human Myeloid Development In Vitro and In Vivo.
Authors: Arseni Natalia , Ahmed Farid , Rawat Vijay P.S. , Sadlon Timothy J. , Hiddemann Wolfgang , Buske Christian , Feuring-Buske Michaela
Published: October 2019

Recent data indicate that a variety of regulatory molecules active in early development may also play a role in the maintenance of hematopoietic stem cells with repopulating activity. Since it was shown, that the Xvent-2 homeobox gene is part of the BMP-4 signalling pathway in Xenopus, we sought to define the expression profile and function of the human homologue VENTX2 in hematopoietic development. When VENTX2 expression was assessed by TaqMan qRT-PCR in highly purified human cord blood subpopulations, expression of VENTX2 was detected in CD33 and CD15 positive myeloid cells as well as in B and T cells. In addition VENTX2 expression was observed in the primitive CD34+38- human subset as well as in 34+38+ compartment. Furthermore, VENTX2 expression was recurrently detected in bone marrow samples from AML patients at diagnosis as determined by TaqMan qRT-PCR (n=3). In an attempt to characterize the functional relevance of VENTX2 expression for hematopoietic development we retrovirally engineered murine hematopoietic progenitor cells to constitutively express the gene using a MSCV-based retroviral construct with an IRES-EGFP cassette. Successfully transduced cells were injected into lethally irradiated mice or used for in vitro experiments. At the level of the clonogenic progenitor VENTX2 induced myeloid and blocked erythroid differentiation with a significant 1.5 fold increase in the number of CFU-G (n=10; p<0.01) compared to the GFP control and a 5.4 fold decrease in the number of erythroid colonies (n=10; p<0.005) without a change of the total number of colonies produced indicating that VENTX2 promoted granulocytic differentiation in vitro. Re-plating assays showed an over 2 fold increase in the number of secondary CFU-G. In MS-5 murine assay VENTX2 transduced cord blood cells increase CD15+ cells 10.4 fold compared to the control (n=3)(p<0,01). When the effect of VENTX2 was determined on the frequency of LTC-IC by limiting dilution assay (n=2), no major differences were detected between the homeobox gene and the control arm. In NOD/SCID mice VENTX2 induced a 2.9 fold increase in the proportion of CD15+ mature myeloid cells as compared to the GFP control (VENTX2 mean 4 x 106 + 1 x 106 n=7; GFP mean 2x106 cells + 6 x 105 n=9; p<0.02). Using competitive repopulation unit assay (CRU) in NOD/SCID mice we could demonstrate that VENTX2 is able to increase the frequency of CRU cells by 1.8 over GFP control (n=4). These findings characterize VENTX2 as a novel regulatory protein of early human hematopoiesis and extend our knowledge on the regulatory role of non-clustered homeobox genes in hematopoietic development.
Phase II Study of Oral Fludarabine (FAMP) in Combination with Rituximab in Indolent B-Cell Non-Hodgkin Lymphoma (B-NHL)
Authors: Ishizawa Kenichi , Tobinai Kensei , Ogura Michinori , Itoh Kuniaki , Morishima Yasuo , Ando Kiyoshi , Yamamoto Joji , Watanabe Takashi , Uchida Toshiki , Nakata Masanobu , Hayashi Masaki , Hotta Tomomitsu
Published: October 2019

Background and objectives: Although intravenous (IV) FAMP in combination with Rituximab was reported to be effective against indolent B-NHL (JCO2005; 23:694), IV administration of FAMP for 3 to 5 consecutive days is inconvenient in an outpatient setting. We conducted a multicenter phase 2 study to evaluate the efficacy and safety of oral FAMP with Rituximab for relapsed or refractory indolent B-NHL. Patients and treatment: Eligible patients were: aged 20 to 74 years, with indolent B-NHL, with measurable lesions (the greatest transverse diameter > 1.5 cm by CT), performance status 0 or 1, with no major organ dysfunctions. Mantle cell lymphoma was excluded. Prior chemotherapies were limited to < = 2 regimens, and prior rituximab treatments up to 16 times were allowed. Patients who received nucleoside analogs or stem cell transplant were excluded. Patients who had progressive disease within 6 months of receiving rituximab therapy were also excluded. Based on the results of the preceding phase 2 study (JCO2006; 24:174), oral FAMP 40 mg/m2 was administered on day 1 to 5, with rituximab 375 mg/m2 on day 1, repeated every 4 weeks, for up to 6 cycles.

Results: Forty-one patients were enrolled and received oral FAMP with rituximab. Thirty-eight patients (93%) were follicular lymphoma, two with MALT lymphoma, and one with small lymphocytic lymphoma. Thirty-four patients (83%) received rituximab with or without chemotherapy prior to enrollment. Median treatment cycles were 6 cycles (range: 2 to 6, 66% of patients completed 6 cycles of treatment). Investigator-assessed overall response rate and complete response rate were 83% (34/41; CI: 68 to 93%) and 76% (31/41; CI: 60 to 83%), respectively. Central evaluation is pending. With median follow-up period of 9 months, median progression-free survival was not reached, and 28 patients (68%) were still alive without disease progression. Toxicity was primary hematologic, transient and manageable. Most common grade 4 hematologic toxicities included lymphopenia (98%), neutropenia (66%), and leukopenia (42%). Grade 3 or greater non-hematologic toxicities were observed in 29% of patients including one each of grade 4 stomatitis and hyperuricemia. Dose reduction of oral FAMP (from 40 to 30 mg/m2) was necessary in 44% of patients at some points of treatment courses. One patient died from Pneumocystis jiroveci pneumonia and other 2 from disease progression after the end of the study.

Conclusion: Oral FAMP combined with rituximab is highly effective with acceptable toxicities for patients with relapsed or refractory indolent B-NHL who have been mostly pretreated with rituximab, and more convenient than the combination with IV FAMP. Prolonged prophylactic therapy against Pneumocystis jiroveci pneumonia would be recommended.
Pre-Treatment p27 and Bcl-6 Staining Levels Correlate with Response to Bortezomib in Non-Hodgkin Lymphoma: Results from a Tissue Microarray Analysis
Authors: Gerecitano John , Gounder Sivaraman , Feldstein Julie , Ogilvie Shahiba , Lin Dorothy , Zheng Junting , Zhang Zhigang , McDonald Alice , Mulligan George , O’Connor Owen A.
Published: October 2019

Background: Proteasome inhibitors (PI) affect a variety of intracellular processes, including the cell cycle, NF-kB and other signal transduction pathways, and the balance of bcl-2-related proteins, yet it remains unknown which of these targets are most crucial in determining response to PI treatment in non-Hodgkin Lymphoma (NHL). Responses to bortezomib (btz) have been seen in different NHL subtypes in several phase 2 studies. The consistency of response suggests that there may be biological characteristics that determine individual tumor responses to PIs. We used tissue microarray (TMA) technology to determine if pre-treatment tumor characteristics can predict outcome of btz treatment in NHL.

Methods: TMAs were constructed using paraffin-embedded blocks of pre-treatment tissue from patients enrolled in a phase 2, CTEP-NCI sponsored trial of btz in NHL. Microsections were stained for 18 proteins with reported relevance to proteasome function and/or lymphoma prognosis. All stains were evaluated and scored by a central pathologist. Staining was correlated with quality and duration of response (DOR) or time to treatment failure (TTF). The best response was treated as a binary outcome (PR/CR vs SD/POD). Correlation between each marker and response was assessed by univariate analysis. The exact Wilcoxon rank-sum test was used to compare DOR and TTF with respect to the binary marker scores.

Results: This analysis includes 35 of 75 patients treated with btz between 11/6/01 and 2/23/07 for whom pre-treatment tissue blocks could be obtained. Response data were available for 32 of these patients. Diagnoses include: FL (N=10), SLL (N=4), MCL (N=14) and MZL (N=4). Expression of the cell-cycle inhibitor p27 was more common in responding vs non-responding pts (p=0.024), and was also significantly associated with longer DOR (p=0.020) and longer TTF (p=0.021; figure). Presence of Bcl-6 was significantly associated with shorter DOR (p=0.049) and shorter TTF (p=0.048; figure), but not response.

Conclusions: Using TMA analysis, we have shown that NHL patients with pre-treatment p27 staining were more likely to respond to btz, and had a longer DOR and TTF than those without baseline staining. Conversely, patients with baseline Bcl-6 expression had a shorter DOR and TTF than those without. Although the number of patients in this analysis is small, the data will help us generate hypotheses about disease and treatment biology that can be tested in larger studies. We are currently collecting samples from a broader pool of patients treated with btz at MSKCC to verify these results. TTF with bortezomib by (A) p27 and (B) Bcl-6 status.

Figure. Figure.
Human B Cell Lines and Primary B Cells Actively Secrete Granzyme B in Response to IL-2 Family Cytokines.
Authors: Jahrsdoerfer Bernd , Blackwell Sue E. , Simmet Thomas , Weiner George J.
Published: October 2019

It is widely believed that the main function of B cells is antibody secretion, but not cellular cytotoxicity. Recently we found that human B cells activated with interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN) develop a phenotype similar to that of cytotoxic T lymphocytes. B cells treated in such a way start to secrete large amounts of granzyme B (GrB) instead of antibodies and, as in the case of B-chronic lymphocytic leukemia (B-CLL), acquire the capability to induce apoptosis in bystander B-CLL cells in a GrB-dependent manner. Using FACS and ELISpot analyses we could now demonstrate that GrB is actively secreted by B cells in a time-dependent manner and that IL-21 is not the only cytokine that induces GrB in B cells. Also cytokine combinations such as IL-10 and IL-4 as well as IL-10 and IFN-alpha induce GrB in normal B cells and various B cell lines including MEC-1 (CLL), ARH-77 (plasma cell leukemia) and Namalwa (Burkitts lymphoma). We conclude that IL-21 and further cytokines can induce B cells to produce functional granzyme B. Further studies are required to elucidate the interactions with B lymphocytes of cells producing these cytokines such as CD4+ T cells, regulatory T cells, NKT cells and plasmacytoid dendritic cells. Our unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity.
Profound Shut-Down of Antigen Specific T-Cell Effector Functions by Dasatinib.
Authors: Weichsel Ralf , Dix Carolin , Wooldrige Linda , Clement Matthew , Fenton-May Angharad , Zezula Josef , Greiner Elisabeth , Gostick Emma , Price David A. , Einsele Hermann , Seggewiss Ruth
Published: October 2019

Dasatinib (Sprycel®, Bristol-Myers Squibb) is a dual BCR-ABL/SRC kinase inhibitor. In 2006, it was approved for the treatment of imatinib-refractory/-resistant CML and Ph+ ALL. An increased infection rate has been described in patients undergoing dasatinib treatment; we postulated that this might be due to inhibition of the SRC kinases LCK and FYN, which play an important role in T cell signalling following antigen recognition. We evaluated dasatinib’s immunobiological effects on purified human CD3+ T cells from healthy blood donors using the promiscuous tyrosine kinase inhibitor staurosporine as a comparator; we also used antigen-specific T cell clones to dissect early T cell signaling events. All assays were performed at clinically relevant doses of dasatinib (1–100nM). In CFSE dilution assays, a dose-dependent inhibition of T cell proliferation was detected with dasatinib (IC50=11nM) and staurosporine (IC50=5nM). These effects of dasatinib, but not staurosporine, were reversible; thus, T cells that were pre-incubated with dasatinib for 24h and then washed, proliferated as well as untreated T cells in the absence of dasatinib. Furthermore, we observed significant inhibition of OKT3-induced up-regulation of the activation marker CD69 (dasatinib IC50=11nM, staurosporine IC50=5nM) and reduced IL-2 production (IC50=2nM, measured by ELISA, for both dasatinib and staurosporine) in purified T cells treated with dasatinib and staurosporine. CD4+ T cells were more sensitive than CD8+ T cells to the inhibitory effects of dasatinib on activation and proliferation (activation IC50=10nM for CD4+ and 15nM for CD8+ T cells; proliferation IC50=10nM for CD4+ and 13nM for CD8+ T cells) while no differences in sensitivity were observed for staurosporine (IC50=4–5 for activation and proliferation in both CD4+ and CD8+ T cells). Since CD8+ T cell-mediated immunity is essential for long-term control of persistent DNA viruses, we evaluated dasatinib’s impact on antigen-specific alpha/beta CD8+ T cell populations specific for CMV and EBV. Profound inhibition of proliferation, cytokine secretion and degranulation was observed in all cases; these observations potentially explain the increased frequency of viral infections in dasatinib-treated patients beyond the induction of myelosuppression. Of note, gamma/delta T cell functions were also inhibited by dasatinib. Dasatinib resulted in significant upregulation of TCR/CD8 at the T cell surface of EBV specific T cell clones indicating that the drug blocks TCR downregulation and recycling at the T cell surface and therefore exerts its effects by blockade of proximal signalling pathways. Overall, these findings provide a rationale to explore the potential of dasatinib as an immunosuppressant in the settings of transplantation and T cell driven autoimmune diseases.