bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.
Authors: Myers Scott N. , Goyal Rakesh K. , Roy Jennifer D. , Ferrell Robert E.
Published: October 2019

Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.
Induction of Growth Inhibition and Apoptosis by TZD18, a Novel Dual Ligand for PPAR alpha/gamma, in Human Ph-Positive ALL Cells In Vitro.
Authors: Elstner Elena , Liu Hongyu , Zang Chuanbing , Liu Dachuan , Xu Shunnan , Fenner Martin H. , Possinger Kurt
Published: October 2019

Peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear hormone receptors which play key roles in the differentiation and lipid metabolism of adipocytes. Recent data frequently indicated that PPAR ligands are also implicated in the growth inhibition, differentiation and apoptosis induction of several human cancers with diverse tissues. We previously showed that Pioglitazone (PGZ), a specific PPARgamma ligand and a member of the approved thiazolidinedione (TZD) class of anti-diabetic drugs, inhibited growth and induced apoptosis of human ALL cell lines including Ph-positive ALL cells (Zang et. al., Leukemia Research, 28:387, 2004). In this study, effects of a novel dual ligand specific for PPARalpha/gamma, TZD18 (MERCK, NJ, USA), on Ph-positive ALL cell lines, BV173, SD1 and Sup-B15 were examined. We noted that treatment of these cells with TZD18 resulted in growth inhibition in a dose-dependent manner which was associated with a G1 to S cell cycle arrest. This growth inhibition was much stronger than that of PGZ. However, this effect seemed not to be meditated through activation of PPARalpha or PPARgamma, since antagonists of PPARalpha or gamma could not reverse it. By studying the key regulators of cell cycle progression, we found that the expression of the cyclin dependent kinase inhibitor (CDKI) p27kip1, but not that of p21cip1, was enhanced whereas the expression of c-myc, cyclin D2, and cyclin dependent kinase 2 and 4 (CDK2 and CDK4) was decreased when these cells were treated with TZD18. Therefore, upregulation of p27kip1 and downregulation of cyclin Ds and CDKs may account for the G1 cell cycle arrest. Furthermore, a remarkable apoptosis induction was found in Ph-positive ALL cells treated with this dual ligand as measured by cell-death ELISA. No obvious alteration of bcl-2 levels but an upregulation of bax were observed in apoptotic cells. An activation of caspase-8 and caspase-9 by this ligand was also noticed. Of clinical importance, TZD18 enhanced the cytotoxic effect of Imatinib, a specific therapeutic agent for Ph-positive ALL. Overall, our findings strongly suggest that TZD18 may offer a new therapeutic agent for treatment of Ph-positive ALL in an adjuvant setting. (This study was supported by grants from Deutsche Jose Carreras Leukaemie-Stiftung and Deutsche Forschungsgemeinschaft to EE)
Leptomycin B Overcomes Imatinib Resistance Mediated by Stromal Cells and Mutant BCR-ABL in Philadelphia Chromosome-Positive Acute Lymphoblastic Leukemia Cells.
Authors: Tojo Arinobu , Izawa Kiyoko , Sekine Rieko , Nagamura Tokiko , Yoshida Minoru , Tsuji Takashi
Published: October 2019

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL) is one of the most intractable hematological malignancies, and readily acquires resistance to chemotherapeutic drugs including imatinib mesylate. We hypothesized that the adhesive interaction of Ph-ALL cells with bone marrow stromal cells might cause their escape from drug-induced apoptosis and subsequent minimal residual disease, resulting in the generation of a chemoresistant clone such as a clone harboring mutant BCR-ABL. To gain insight into this possibility and a novel strategy against imatinib resistance, we used two Ph-ALL cell lines designated as IMS-PhL1 (L1) and IMS-PhL2 (L2). L1 cells had wild type BCR-ABL, whereas L2 cells had Y253H mutant and revealed 10-fold or more resistance to imatinib, compared with L1 cells. The growth of L1 cells was autonomous and their spontaneous apoptosis was suppressed by co-culture with a murine bone marrow stromal cell line, HESS-5. In contrast, the sustained growth and survival of L2 cells was absolutely dependent on direct contact with HESS-5. Both cell lines adhered to and migrated beneath the HESS-5 cell layer, resulting in the formation of cobblestone areas (CA). While floating L1 cells were eradicated by 1 mM imatinib, a portion of adherent L1 cells could survive even at 10 μM imatinib. Similarly, L2 cells forming CA beneath the HESS-5 cell layer considerably resisted prolonged exposure to 10 μM imatinib. Leptomycin B (LMB), a potent inhibitor of CRM1/exportin-1, can trap BCR-ABL in the nucleus and can aggressively eliminate BCR-ABL+ cells in combination with imatinib (Wang et al., 2001). We tested LMB for its ability to eliminate CA or adherent Ph-ALL cells in combination with imatinb. The result for L2 cells was shown in Figure. Dramatically, combined use of 10 μM imatinib and 1 nM LMB for 7 days exerted a synergistic effect on reduction in the number of CA. L1 cells were also susceptible to the combination of imatinib and LMB. Our results suggest that nuclear entrapment of BCR-ABL may be a promising strategy for overcoming imatinib resistance mediated by stromal cells as well as a certain BCR-ABL mutant.

Figure Figure
Synergism between 17-AAG and Imatinib in Imatinib-Resistant CML Cells: Inhibition of P-Glycoprotein by 17-AAG as a New Mechanism of Increasing Imatinib Activity.
Authors: Radujkovic Aleksandar , Schad Moritz , Topaly Julian , Laufs Stephanie , Jauch Anna , Schultheis Beate , Melo Junia V. , Ho Anthony D. , Fruehauf Stefan , Zeller W. Jens
Published: October 2019

Chronic myelogenous leukemia (CML) has gained outstanding importance for targeted cancer therapy. Inhibition of the BCR-ABL tyrosine kinase by imatinib (STI571, Glivec, Gleevec) leads to reduced proliferation of CML cells in vitro and sustained hematological and cytogenetic responses in vivo. However, resistance has been observed after variable periods of imatinib monotherapy especially in advanced stages of disease and overexpression of the BCR-ABL protein is one of the mechanisms of imatinib resistance. As combination therapy may allow to overcome drug resistance, we were interested in the effect of combination treatment with imatinib and 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the heat shock protein 90 (Hsp90) chaperone complex. Furthermore, a new mechanism of action of the heat Hsp90 inhibitor 17-AAG is brought to light giving implications for an additional benefit of a combination treatment of imatinb-resistant chronic myelogenous leukemia. In imatinib-sensitive CML cell lines, combination index values (CI) obtained using the method of Chou and Talalay indicated additive (CI = 1) or slightly antagonistic (CI > 1) effects following simultaneous treatment with imatinib and 17-AAG. In contrast, the agents acted synergistically in imatinib-resistant BCR-ABL overexpressing LAMA84-R cells (CI = 0.6 at 75 % growth inhibition level). Growth inhibition of CFU-GM colonies of primary CML cells obtained from 3 patients is stronger after combination treatment than after monotherapy and annexin V / propidium iodide staining showed a strong increase of the apoptotic cell fraction in CML cells treated for 48 hrs by the combination treatment as compared to treatment with each drug alone. In imatinib-resistant cells BCR-ABL mRNA levels and protein expression were increased compared to the imatinib-sensitive parental cell line which is consistent to our cytogenetic and multicolor FISH analyses revealing multiple genomic BCR-ABL copies in imatinib-resistant cells. Furthermore, in imatinib-resistant cells P-glycoprotein activity was increased. Single treatment with 17-AAG lowered BCR-ABL and increased Hsp70 protein levels in both cell lines as expected whereas combination treatment was even more effective in this respect. Interestingly, single treatment with 17-AAG also decreased P-glycoprotein activity in a dose-dependent fashion as confirmed by a rhodamine-123 exclusion assay. The synergistic effect of both drugs in imatinib-resistant cells may thus be explained by increased intracellular levels of imatinib following 17-AAG treatment. The relevance of this additional mechanism warrants further exploration in clinical studies.
Zoledronate Inhibits Leukemia Growth in Bone Marrow and Synergizes with Imatinib Mesylate Against Ph+ Primary Leukemic Cells.
Authors: Segawa Hidekazu , Kimura Shinya , Kuroda Junya , Yuasa Takeshi , Maekawa Taira
Published: October 2019

Imatinib mesylate has drastically changed Philadelphia chromosome positive (Ph+) leukemia treatment. However, remissions induced in advanced phase Ph+ leukemias tend to be short-lived even treated with imatinib. Combined therapy of imatinib with an agent which inhibits downstream signaling of BCR/ABL such as Ras is intriguing. Phase I studies of farnesyl transferase inhibitors (FTIs), which targeted farnesylation of Ras, with imatinib against Ph+ leukemias demonstrated limited combination effects. We previously reported that the third-generation bisphosphonates (BPs) zoledronate (ZOL) synergized with imatinib mesylate to inhibit Ph+ leukemia. BPs which inhibit both farnesylation and geranylgeranylation of Ras proteins may be more potent than FTIs. However, three important questions have remained unanswered to promote this attractive combination to a clinical trial. Concentrations of 20-30 μM ZOL are required in vitro to induce apoptosis in leukemic cell lines. However, peak serum concentrations after infusion of 4 mg ZOL were 1–2 μM. Therefore, it is important to investigate how ZOL achieves effective concentrations in vivo. The second is whether ZOL augments the effects of imatinib against not only cell lines but also primary cells from Ph+ leukemia patients. In addition, the safety of this combination therapy must be established, because the dose of ZOL used in our previous study was high compared with clinical doses administered for inhibition of bone lesions. To answer these questions, we herein investigated about the combination therapy of ZOL plus imatinib using NOD/SCID mice engrafted with primary Ph+ leukemic cells with or without mutations in the ATP binding site. ZOL inhibited the geranylgeranylation of Ras proteins of leukemic cells in bone marrow but not in peripheral blood, indicating that sufficient concentration of ZOL could be achieved in bone marrow. ZOL synergized with imatinib to enhance survival of mice engrafted with primary Ph+ leukemic cells without mutations and this combination was well-tolerated without any severe adverse effects. These findings suggest that ZOL inhibits leukemia growth in bone marrow and the combination of ZOL plus imatinib is a potential therapy for Ph+ leukemia patients.
Investigating the Biological and Molecular Mechanisms Underpinning the Engraftment/Selection Advantage Conferred to Hematopoietic Stem Cells by HOXB4.
Authors: Milsom Michael D. , Hollins Laura , Gagen Dorothy , Woolford Lorna B. , Fairbairn Leslie J.
Published: October 2019

We have recently demonstrated that co-expression of HOXB4 enables the enhanced delivery of HSC harbouring a second therapeutic trans-gene. Nonetheless, it is of great importance to elaborate the current knowledge about the mechanism of HOXB4 action in order to both evaluate the safety implications of its use in a clinical strategy, and to gain greater insight into the regulation of HSC self-renewal/expansion. To these ends we have performed an extensive in vitro analysis of the consequences of HOXB4 overexpression in primary murine BMC and in a murine multipotent myeloid progenitor cell line (FDCP-mix). We demonstrate for the first time in murine cells, that ectopic HOXB4 reduces the responsiveness of murine hematopoietic cells to differentiation stimuli. Furthermore, by performing a detailed investigation into the kinetics of FDCP-mix differentiation, we reveal that HOXB4 overexpression results in a specific differentiation delay as opposed to an outright block. We propose that an analogous delay is in operation in repopulating cells in order that the shift to increased assymetrical self-renewal, a requirement for stem cell expansion, is achieved. Notwithstanding this, it is clear that any perturbation in differentiation constitutes an increased risk of cellular transformation if this technology were transferred to a clinical setting. In order to further define the repercussions of ectopic HOXB4 delivery, we have developed a retroviral vector which encodes an activatable version of HOXB4. We have shown that this vector is able to mediate an in vitro differentiation delay in primary murine BMC and FDCP-mix as well as enable enhanced engraftment of BMC in vivo, both dependent upon the addition of the estrogen analogue; tamoxifen. Using this system, we are currently examining the effect of ectopic HOXB4 on the transcriptome of FDCP-mix cells, in addition to performing an in depth study into the biological mechanisms affected by HOXB4 overexpression in BMC in vivo. We envisage that these model systems will be particularly amenable to the manipulation required for target gene identification/validation.
Purification of Allogeneic Idiotype-Specific T Lymphocytes According to IFN-γ Production for Adoptive Immunotherapy in Multiple Myeloma Patients.
Authors: Curti Antonio , Pandolfi Simona , Isidori Alessandro , Baccarani Michele , Lemoli Roberto M.
Published: October 2019

In multiple myeloma (MM) adoptive immunotherapy through allogeneic donor lymphocyte infusions results in a well-documented graft versus myeloma effect, but it is associated with high incidence of graft versus host disease. The idiotype (Id) expressed by MM cells can be regarded as a tumor-specific antigen and it has been used for immunotherapy. To enhance antitumor immunity and reduce alloreactivity, allogeneic T cells were activated against tumor-derived Id and then purified according to IFN-γ production. Total peripheral blood mononuclear cells from healthy donors were incubated with autologous monocyte-derived dendritic cells and pulsed with patient-derived Id protein. Cells were maintained in serum-free medium and supplemented during the priming phase with IL-7 and IL-12. Subsequently, the T-cell culture was restimulated every 7 days with pulsed DCs in the presence of low doses of IL-2. After 2 or 3 stimulations, the percentage of IFN-γ-producing T cells was as high as 5–10%, whereas that observed in presence of not stimulated T-cells was undetectable. Based on their IFN-γ production, T cells were isolated by using a commercial immunomagnetic IFN-γ capture assay. The purity of enriched IFN-γ-producing T cells ranged between 60 and 90% as evaluated by flow cytometry. The yield was 60% of pre-selection IFN-γ positive T cells and cell viability after selection was 80%. Functionally, IFN-γ purified T cells showed better Id-specific proliferative response when compared both to not-stimulated T cells and to stimulated but not purified T cells. IFN-γ purified T cells were shown to be memory CD45RO+ T cells expressing HLA-DR, CD25, CD95 at high level, whereas the negative fraction was mainly composed of naive, poorly activated T cells. T-cell receptor spectratyping analysis demonstrated that IFN-γ positive T cells were represented by a pauciclonal cell population as compared to policlonal T cells obtained before cell culture. Moreover, addition of not-stimulated T cells resulted in a significant reduction of allogeneic CD34+-derived colony-forming capacity. Conversely, the absolute number of allogeneic total colony-forming units-cells was not affected by IFN-γ purified T cells.These data demonstrate that Id-specific T cells may be generated from healthy donors and significantly enriched on the basis of their IFN-γ production. Id-specific IFN-γ - purified T cells have better anti-Id response and reduced alloreactivity than unselected T cells.
Allogeneic Stem Cell Transplantation with Reduced-Intensity, Fludarabine-Based Conditioning in Relapsed and Refractory Hodgkin’s Disease: Low Transplant-Related Mortality and Impact of Intensity of Conditioning Regimen on Survival.
Authors: Anderlini Paolo , Saliba Rima , Donato Michele , Giralt Sergio , Andersson Borje , Ueno Nato T. , Khouri Issa F. , de Lima Marcos , Ippoliti Cindy , Champlin Richard E.
Published: October 2019

Forty patients with relapsed or refractory Hodgkin’s disease (HD) underwent allogeneic stem cell transplantation (allo-SCT) following a fludarabine-based conditioning regimen from an HLA-identical sibling (n=20) or a matched unrelated donor (n=20). The median age was 31 years (range 18-58). The median number of chemotherapy regimens received prior to allo-SCT was five (range 2-9). Thirty (75%) and thirty (75%) patients had received prior radiotherapy or a prior autologous SCT, respectively. The median time to progression after autologous SCT was nine months (3–52). Disease status at SCT was refractory relapse (n=14) or sensitive relapse (n=26). The conditioning regimens employed were fludarabine (25 mg/m sq IV x 5 days)-cyclophosphamide (1 g/m sq IV x 3 days) ± antithymocyte globulin (30 mg/kg IV x 3 days) (FC±ATG) (n=14), a less intensive regimen, and fludarabine (25 mg/m sq IV x 5 days) -melphalan (70 mg/m sq IV x 2 days) (FM) (n=26), a more intensive one. The two groups had similar demographics and prognostic factors. Chimerism studies indicated 100% donor-derived engraftment in 26/26 (100%) FM patients and in 9/13 (69%) evaluable FC±ATG patients. Day 100 and cumulative (18-month) transplant-related mortality (TRM) were 5 % and 22%, respectively for the whole group. There was a nonsignificant trend towards a lower cumulative TRM in the FM group (18% vs. 30% at 18 months, p=0.2). The cumulative incidence of acute (grade II-IV) GVHD was 38%. The cumulative incidence of chronic GVHD at 18 months was 69%. There was a trend for a lower relapse rate after the occurrence of GVHD, however, this was not statistically significant (hazard ratio 0.8; p= 0.6). Progression rates were similar in the FM and FC patients (53% vs. 57% respectively at 18 months, p=0.4). However, disease progression occurred later in FM patients (range 2–34 months) than in FC patients (range 0.7–13 months). In addition, with comparable follow-up time after progression, the FM group experienced a lower death rate after progression. Twenty-four patients (60%) are alive (fourteen in complete remission) with a median follow-up of 13 months (4–78). Sixteen patients expired (TRM n=8, disease progression n=8). FM patients had significantly better overall survival (73% vs. 39% at 18 months; p=0.03), and a trend towards better progression-free survival (37% vs. 21% at 18 months; p=0.2). We conclude that allo-SCT with fludarabine-based, less intensive conditioning from matched related and unrelated donors are feasible in high-risk HD patients with a low TRM. The intensity of the preparative regimen affects survival.
Minor Histocompatibility Antigen-Specific CD8+ Memory T Cells in Hematopoietic Compartments Survive the Immediate Non-Ablative and Ablative TBI Milieu.
Authors: Shatry Alwi M. , Levy Robert B.
Published: October 2019

Minor histocompatibility (MiHA) antigens induce CD8+ T-cell responses that mediate resistance to bone marrow engraftment in an MHC-identical, MiHA-disparate marrow transplant model, in which recipients are sensitized to donor antigens (BALB.B à B6BALB.B) prior to BMT. The H60 H antigen has been shown to dominate the immune response in B6 mice primed with BALB.B antigens (B6BALB.B). We initially sought to determine the distribution and partially characterize the phenotype of H60-specific CD8+ cells in the blood, spleen and marrow compartments of B6 mice primed with 3 x 107 BALB.B lymphoid cells and ≥3 weeks later boosted with 2 x 107 cells. An H60 tetramer (LTFNYRNL/H2-Kb) conjugated to PE was used to detect H60-specific CD8+ cells in these compartments. Eight days following the second immunization, the mean frequency of circulating H60-specific cells was 12.2% ± SE 0.88 of CD8+ cells (range: 5.6 – 20.5%). The frequency of splenic H60-specific CD8+ cells was equivalent to that of circulating antigen-specific cells, thus peripheral blood levels of H60-specific CD8+ cells appear to be representative of those resident in the spleen. Interestingly, in the marrow compartment, the frequency of H-60+ cells amongst the CD8 T cell population was higher compared to peripheral blood and spleen levels, suggesting that H60-specific CD8+ cells in this compartment may comprise both migrant cells from the periphery and resident cells in the marrow elicited during priming. In both BM and spleen, >90% of CD8+ H60+ cells expressed the memory phenotype(CD44+, Ly6C+) and as expected, did not express early activation markers (CD25, CD69). To mediate resistance to progenitor cell engraftment, H60-specific effector CD8+ cell must first survive the immediate post-BMT milieu in the hemopoietic compartments. To examine this question, B6BALB.B mice irradiated at 3.0, 6.0 and 9.0 Gy were analyzed 24 hours later for the presence of H60-specific CD8+ cells in the spleen and marrow compartments. Although there was an expected irradiation dose-dependent decrease in absolute numbers of CD8+ H60+ cells in the two compartments, there was a dose-dependent increase in percent of CD8+ T cells expressing the H60 TCR in both compartments. This observation indicates enhanced survival of these antigen-specific CD8+ memory T cells post-conditioning. Preliminary results indicate that 24h post-BMT into 9.0 Gy TBI recipients, there was BrDU uptake in marrow and splenic CD8+H60+ T cells in B6BALB.B transplanted with 1 x 107 BALB.B BM-TCD. Approximately 80% of CD8+H60+ T cells in the marrow compartment of primed recipients of BALB.B cells exhibited proliferation by BrDU uptake. Thus, donor MiHA-disparate marrow grafts elicit antigen-driven proliferation early post-BMT by CD8+ memory T cells in both compartments consistent with the potential importance of these cells in mediating resistance against progenitor engraftment across these MiHA differences.
Acute Graft-Versus-Host Disease: Clinical Presentation and Response to Therapy Following Umbilical Cord Blood Transplant.
Authors: Arora Mukta , Wagner John E. , Barker Juliet N. , Miller Jeffrey S. , Burns Linda J. , MacMillan Margaret L. , Blazar Bruce R. , Defor Todd E. , Weisdorf Daniel J.
Published: October 2019

The nature of aGVHD after umbilical cord blood transplant (UCBT) has not been well characterized. We analyzed acute graft-versus-host disease (aGVHD) incidence and response to therapy in 136 recipients of unrelated UCBT. aGVHD developed in 72 receiving myeloablative (MA) (n=39) and non ablative stem cell transplant conditioning (NST) (n=33) (indicated if age >45 years, prior transplant and major co-morbidity). MA transplant recipients were significantly younger (median age 14 years, range 0.8–50) than NST transplant recipients (median 48 years, range 0.7–59), p < 0.0001 and had fewer advanced malignant disease at transplant (8% versus 24%), p = 0.09. At diagnosis of aGVHD, the clinical grades and organ involvement were similar in the 2 groups. Grade II–IV aGVHD developed in 34% of MA transplant recipients and 45% (p = 0.1) of NST transplant recipients at a median of 35 days (12–137) and 30 days (15–96) post transplant whereas grade III–IV aGVHD developed in 9% and 13% (p=0.6), respectively. 87% of MA vs. 85% of NST recipients (p=0.8) had skin involvement; 39% vs. 49% had GI disease (p=0.4); and 10% vs.9% (p=0.9) had liver GVHD, respectively. Response to therapy was assessed at 28 and 56 days after diagnosis of aGVHD. A high frequency of complete response (CR) was seen across all grades at day 28 (47%, 48% and 43% CR in grades I, II and III–IV, respectively). At day 56, CR was more frequent in grade I aGVHD (68% vs 64% and 50% in patients with grade II and III–IV disease, respectively, p 0.08). Response rates were similar in MA and NST transplant recipients (CR in 49% vs 46% by day 28, p=0.1, and 64% vs 61% by day 56, p=0.5. Age < 20 years (CR or PR 82% vs 64%, p = 0.09) and single organ GVHD (81% vs 52%, p=0.01) were associated with a higher response. A higher response rate was seen in patients with skin GVHD (85%) vs GI disease (63%) vs skin + GI (56%) vs liver involvement with skin/GI (43%), p=0.04. After a median follow up of 1.0 year (0.8–7 years) in both groups, 67% (95% CI 52%–81%) of MA versus 45% (95% CI 28%–62%) of NST patients survive (p=0.05). Higher 1 year survival of 70% (95% CI 58%–83%) was seen in patients with day 28 CR or PR to treatment versus non responders (24%, 95% CI 6%–42%), p<0.0001.

This data suggests that among unrelated UCBT recipients using either MA or NST preparative regimens, similar incidence, clinical presentation and response to therapy of aGVHD was seen. Early identification of high risk group (age >20, multiorgan disease, GI or liver GVHD) should facilitate timely assignment of intensified therapy.
Favorable Outcome for Infant ALL Following Hematopoietic Stem Cell Transplantation (HSCT).
Authors: Jacobsohn David A. , Hewlett Brad , Morgan Elaine , Duerst Reggie E. , Kletzel Morris
Published: October 2019

There exists controversy regarding the benefit of HSCT in infant ALL. At our institution, the standard treatment for high-risk infant ALL has been intensive chemotherapy per POG 9407 followed by HSCT in CR1. We retrospectively reviewed the outcome of the 15 patients (9 males, 6 females) with infant ALL that underwent allogeneic HSCT since 1992. We defined “very high-risk” as an infant with one or more of the following criteria: age 6 months or less at diagnosis, MLL+ or t(4:11), or WBC>100,000 at diagnosis. Fourteen of fifteen patients are considered “very high-risk.” All except one were under 12 months at diagnosis (range 2–14 months, median 7). The fourteen month old was included as she displayed infant ALL biology, with MLL+ and WBC>500,000. Seven were 6 months or under at diagnosis. Cytogenetics at diagnosis showed MLL+ or t(4:11) in 9, normal in 4, and indeterminate in 2 patients. WBC count at diagnosis was 4,300–1,000,000 (median 360,000). Twelve of the patients were CALLA-negative and four had CNS involvement at diagnosis. Time from diagnosis to transplant was 2.3–13.6 months (median 4.2). All patients were in morphologic CR1 at time of HSCT but one patient had persistent t(4:11).

Cytoreduction: Patients received TBI 150cGy x 8 (d-10 to-7); VP-16 1g/m2 CI (d-5 to-4); cyclophosphamide 60 mg/kg/day x 3(d-4 to-2). GVHD prophylaxis: CSA, short-course MTX, and ATG d +1, +3, +5, +7 (for unrelated cord blood (UCB)). Grafts were not T-cell depleted.

Stem cell sources: 8 UCB, 6 HLA-identical sibling bone marrow, 1 HLA-identical sibling PBSC. Days to neutrophil engraftment was 11–25 days (median 16) and to platelet engraftment was 17–82 days (median 37). CNS prophylaxis consisted of intrathecal methotrexate and Ara-C monthly x 6 post-HSCT. Acute GVHD: 2 grade II and 1 grade III. Chronic GVHD: 3 limited, 1 extensive.

Mortality: 4 patients have died. 2 (13%) of transplant-related mortality (TRM) within 100 days of HSCT and 2 of relapse (15% of evaluable patients), which included the patient with t(4:11) at time of HSCT. Range of follow-up is 0.1–11.8 years with a 73% event-free survival (EFS) and overall-survival at a median follow-up of 3.5 years. We conclude that patients with infant ALL who attain a CR1 (morphologic and cytogenetic) have good EFS with acceptable TRM following HSCT using either UCB or HSC from a matched-sibling. Long-term follow-up, including developmental assessments, are being pursued to document the impact of this intensive therapy in young children.

Figure Figure
Outcomes of Transplantation with Matched-Related Donor (MRD) and Alternative Donor (AD) Stem Cells in Children with Severe Thalassemia.
Authors: Hongeng Suradej , Pakakasama Samart , Chuansumrit Ampaiwan , Sirachainan Nongnuch , Chaisiripoomkere Watana , Ungkanont Artit , Jootar Saengsuree
Published: October 2019

Allogeneic stem cell transplant (SCT) from an HLA identical family donor is an accepted option for severe thalassemia. However, the availability of MRD is only 30%. Therefore, AD is another option. The aim of this study is to explore the outcomes of allogeneic SCT with MRD and AD in our institution. We studied 46 consecutive patients (pts) who received MRD (n = 24) or AD; matched unrelated and haploidentical donors (n = 22) stem cells (SCs) for severe thalassemia between September 1992 and July 2004. In MRD group, 3 pts received 1 Ag HLA-mismatched SCs and in AD group, 2 pts received haploidentical SCs. The characteristics of both groups were followed.

The conditioning regimen consisted with busulfan 16 mg/kg, cyclophosphamide 200 mg/kg for MRD and additional ALG (Fresineus) 40 mg/kg for matched unrelated donor (MUD) and CBT and busulfan 8–12 mg/kg, fludarabine 175 mg/m2, ALG, + thiotepa, and + total lymphoid irradiation for NST. GVHD prophylaxis was cyclosporin and MTX for MRD, FK 506 and MTX or MMF for MUD, cyclosporin, and prednisolone for CBT, and FK 506 and MMF for NST. The 2 yr thalassemia free survival rate for MRD is 83% and AD 81% (p = 0.6). The 2 yr overall survival rate for MRD is 95% and AD 90 % (p = 0.5). Two (8%) pts of MRD and 2 (9%) of AD had severe acute GVHD grade 3–4. Three pts of MRD had chronic GVHD (2;limited, 1;extensive). Three pts of AD had limited chronic GVHD. Rejection rate of MRD group is 12.5% (n=3). Two of 3 MRD pts who had graft failure after first transplant were successful for second transplant. Rejection rate of AD group is 9% (n=2). In conclusion, the outcomes of transplantation with MRD and AD stem cells in children with severe thalassemia are both equally favorable. Further study should be investigated in a larger group of patients to confirm our findings.

MRD (n = 24) AD (n = 22) Age (yr) (median) 2-19 (4) 1-17 (7) β thalassemia major 8 9 β thalassemia / HbE 16 13 Lucarelli class I 12 16 Lucarelli class II and III 12 6 Bone Marrow Transplant (BMT) 11 19 Peripheral Blood Stem Cell Transplant (PBSCT) 8 – Cord Blood Transplant (CBT) 3 1 Nonmyeloablative Stem Cell Transplant (NST) 2 2
Flt3 Receptor Expression Rescues Dendritic Cell Developmental Potential in Megakaryocyte/Erythrocyte Progenitor Cells.
Authors: Onai Nobuyuki , Onai Aya , Tussiwand Roxane , Lanzavecchia Antonio , Manz Markus G.
Published: October 2019

Flt3/Flt3-ligand has a non-redundant role in dendritic cell (DC) and interferon producing cell (IPC) development from hematopoietic progenitor cells. Flt3-ligand−/− mice display a severe reduction of DCs and IPCs, and both, Flt3-ligand injection and conditional over- expression of Flt3-ligand in mice leads to an increased pool size of both cellular subsets. Furthermore, DC and IPC developmental precursor activity segregates with Flt3+ progenitor populations in mouse bone marrow. We have reported that common myeloid progenitors (CMPs) and their progeny granulocyte/macrophage progenitors (GMPs) as well as common lymphoid progenitors (CLPs) and pro-T cells can give rise to DCs in vitro and in vivo. DC developmental potential was lost, however, once definitive megakaryocyte/erythrocyte or B cell commitment occurs, both steps that are accompanied by loss flt3 receptor expression. To test if DC/IPC developmental potential can be regained, we evaluated the effect of ecotropic expression of human Flt3 in lin−c-kit+Flt3−Thy1.1−IL-7Ra− cells (flt3− progenitors) and in megakaryocyte/erythrocyte progenitors (MEPs).

hFlt3 was transduced into purified flt3− progenitors, MEPs, and GMPs using a retrovirus vector carrying both hFlt3 and GFP. The transduction efficiencies were 20~25%. hFlt3-transduced flt3− progenitors gave rise to functional DCs and IPCs, but not control-GFP-transduced flt3− progenitors in vitro. Similarly, hFlt3-transduced MEPs differentiated into functional DCs in Flt3L, SCF, GM-CSF, IL-4, and TNF-a supplemented cultures but not into TER119+ erythroid cells. DC developmental potential of hFlt3-transduced MEPs was comparable to GFP-transduced GMPs. Over expression of hFlt3 in GMPs resulted in augmentation of its DC developmental potential. Interestingly, both hFlt3-tarnsduced MEPs and GMPs preferentially differentiated into IPCs in Flt3L supplemented medium. RT-PCR analysis revealed that enforced Flt3 signaling in MEPs lead to up-regulation of GM- and/or DC-development affiliated genes such as G-CSF receptor, M-CSF receptor, and GM-CSF receptor, PU.1, C/EBPa and STAT3. Furthermore, hFlt3-transduced MEPs gave rise to myeloid colonies (CFU-GM/G/M) but not CFU-Mix colonies in GM-CSF, IL-3, SCF, Flt3L, Tpo, and Epo supplemented methylcellulose medium. These results show that artificial Flt3 signaling in otherwise Flt3− progenitors is sufficient to reestablish DC/IPC differentiation and GM-differentiation programs.
Involvement of Anti-Apoptotic Signaling in Flt3-Dependent B Lymphopoiesis.
Authors: Jensen Christina T. , Sasaki Yutaka , Sitnicka Ewa , Jacobsen Sten Eirik W.
Published: October 2019

Cytokine signaling through the tyrosine kinase receptor flt3 and the hematopoietin Interleukin-7-receptor a chain (IL7Ra) is critically involved in regulation of B lymphopoiesis. Specifically, we have recently demonstrated that mice double deficient in flt3 and IL7Ra signaling fail to support B cell commitment and development during embryogenesis as well as in adult hematopietic tissues (Sitnicka et al, J. Exp. Med; 198:1495, 2003). Previous studies have through generation of IL7Ra knockout mouse on a Bcl-2 transgenic background suggested that IL-7 induced triggering of anti-apoptotic pathways is critically involved in T cell but not B cell development (Akashi et al, Cell; 89:1033, 1997, Kondo et al, Immunity; 7:155, 1997). Thus, we here investigated whether anti-apoptotic signaling might rather be involved in flt3-dependent B cell development, through generation of a H2k driven human Bcl-2 transgenic/Flt3 ligand (FL)−/− (Bcl-2/FL−/−) mice. Strikingly, numbers of Common Lymphoid Progenitors (CLP; lin−c-kitloSca-1loIL7Ra+), pro-B (B220+CD19+AA4.1+CD43+), pre-B (B220+CD43−IgM−) and mature B cells (B220+CD43−IgM+) in the BM of 7–8 weeks old Bcl-2/FL−/− mice were all comparable to those observed in wild type mice. More importantly, the pronounced reductions in CLP, pro-B and pre-B progenitors seen in FL−/− mice on a wild type background were compensated by approximately 50% when FL-deficiency was rather induced on a bcl-2 transgenic background (Bcl-2/FL−/−). Thus, in contrast to IL7Ra, the critical role of flt3 in B cell commitment and development might involve triggering of anti-apoptotic pathways.
Development of a Disease-Specific Utility Instrument for Hemophilia.
Authors: Wasserman Joan E. , Hoots William K.
Published: October 2019

Research has shown that disease-specific health-related quality of life (HRQoL) instruments are more responsive than generic instruments to particular disease conditions. However, only a few studies have used disease-specific instruments to measure HRQoL in hemophilia. The goal of this project was to develop a disease-specific utility instrument that measures patient preferences for various hemophilia health states. The visual analog scale (VAS), a ranking method, and the standard gamble (SG), a choice-based method incorporating risk, were used to measure patient preferences. Study participants (n=128) were recruited from the UT/Gulf States Hemophilia and Thrombophilia Center and stratified by age: 0 – 18 years and 19 +. Test retest reliability was demonstrated for both VAS and SG instruments, overall within-subject correlation coefficients were 0.91 and 0.79, respectively. Results showed statistically significant differences in responses between pediatric and adult participants when using the SG (p=.045). However, no significant differences were shown between these groups when using the VAS (p=.636). When responses to VAS and SG instruments were compared, statistically significant differences in both pediatric (p<.0001) and adult (p<.0001) groups were observed. Stratification of the study sample into mild, moderate and severe disease categories yielded no statistically significant differences in patient preference values among the three groups for both instruments (VAS (p =0.578 for mild vs. moderate, p =0.590 for mild vs. severe and p= 0.920 for moderate vs. severe and SG (p= 0.578 for mild vs. moderate, p = 0.590 for mild vs. severe and p=0.920 for moderate vs. severe). The utility measures obtained from this study can be applied in economic evaluations and decision models that analyze the cost/utility of alternative hemophilia treatments. Results derived from the SG indicate that age can influence patients’ preferences regarding their state of health. This may have implications for considering treatment options based on the mean age of the population under consideration. Data from this study also demonstrated that persons with varying severity of disease were able to objectively evaluate a wide range of health states for hemophilia. When assessing HRQoL for a rare disease such as hemophilia, it is important to establish that HRQoL measurements can be applied broadly across the population. Although the VAS and SG independently demonstrated reliability and validity, results indicate that the two measures may not be interchangeable.
Highly-Purified Aspergillus Fumigatus Specific T-Cells as a Potential Treatment Option of Invasive Aspergillosis.
Authors: Beck Olaf R. , Topp Max S. , Koehl Ulrike , Hanisch Mitra , Hebart Holger F. , Latge Jean-Paul , Einsele Hermann , Klingebiel Thomas , Lehrnbecher Thomas
Published: October 2019

Invasive aspergillosis is still a life-threatening complication, in particular in patients after allogeneic hematopoietic stem cell transplantation (HSCT). Whereas prolonged neutropenia is a well established risk factor for invasive fungal disease, there is a growing body of evidence that T-cells also play an important role in the immunological response to Aspergillus species. Since invasive aspergillosis often occurs during the phase of postengraftment, which is characterized by impaired cell-mediated immunity, Aspergillus-specific T-cells could be a potential therapeutic target in these patients. We therefore analyzed in a first step the response of T-cells to several potential antigens of Aspergillus fumigatus by means of 3H-thymidine incorporation assay. In order to generate Aspergillus-specific T-cells, the antigens with the highest proliferation indices (EC-SAB and 90 kDa catalase) were used to stimulate 1.0 x 108 mononuclear cells from healthy donors. The activated T-cells were isolated on the following day using the IFN-γ secretion assay (Miltenyi Biotec, Germany) and then expanded for 14 days. Intracellular cytokine analysis of EC-SAB generated cell lines (n=7) revealed a significant IFN-γ secretion by 13.6%±2.3 of CD4+ cells (seven out of seven tested cell lines) and an Aspergillus-specific IL-2 secretion by 6.5%±1.9 of CD4+ cells (three out of three tested cell lines), which supports the TH1 response of the generated cells to Aspergillus antigen. In contrast to EC-SAB generated T-cell populations, all three cell lines which were generated with 90 kDa catalase were not informative. Further analysis showed that restimulation with EC-SAB induced a strong proliferation of EC-SAB generated T-cell populations (all three populations tested), whereas alloreactivity was unaffected. The number of these cells could be expanded within 14 days up to 20fold using OKT-3, IL-2 and feeder cells. Currently, we investigate the impact of these Aspergillus-specific cell populations in the defense to different species of Aspergillus. Our preliminary results suggest that Aspergillus-specific T-cells could be an interesting option in prophylaxis and therapy of invasive aspergillosis in patients undergoing HSCT.
Pamidronate Therapy for One Year after Allogeneic Bone Marrow Transplantation (AlloBMT) Reduces Bone Loss from the Lumbar Spine, Femoral Neck and Total Hip.
Authors: Grigg Andrew C. , Shuttleworth Peter , Reynolds John , Szer Jeff , Schwarer Anthony P. , Roberts Andrew W. , Bradstock Ken F. , Hui Chi-Hung , Herrmann Richard , Ebeling Peter
Published: October 2019

We and others have demonstrated that substantial loss of bone mineral density (BMD) is very common following alloBMT, in part due to prolonged use of glucocorticoids. Intravenous (IV) pamidronate is a potent bisphosphonate with efficacy in preventing glucocorticoid-induced bone loss in the non-BMT setting. 116 alloBMT recipients were randomised at 5 institutions to receive pamidronate 90mg IV monthly from day-7 to one year post-transplant (n=63) or no pamidronate (n=53) in an open label, prospective, controlled trial. All patients received oral vitamin D and calcium supplements and all women also received hormone therapy with an oestrogen and progestin. The primary end-point was the reduction in bone loss from the lumbar spine, femoral neck and total hip at 12 months post BMT. Age, sex and conditioning regimen (total body irradiation versus chemotherapy only) were not significantly different between the groups. 37 patients were not evaluable, predominantly due to early death (n=29) or protocol violations (n=4). Significant reductions in BMD loss at 12 months were seen in all 3 evaluated sites in patients treated with pamidronate.

Site No pamidronate Pamidronate p value n % change* n % change* *percentage change in BMD at 12 months Lumbar Spine 28 −3.77 46 2.53 <.0001 Femoral neck 27 −9.33 45 2.82 <.0001 Total Hip 23 −8.35 38 −3.32 .0072

There were no significant differences in the 12-month changes associated with age class (<30, 30–40, 40–50, >50 years) or sex. In preliminary analyses of steroid dose subgroups, statistically significant improvements in BMD loss were found in patients whose average daily equivalent prednisolone dosage in the first six months post transplant was i) >25mg for all sites and ii) 10–25mg daily for the lumbar spine. We conclude that prophylactic pamidronate significantly reduces bone loss from the spine and hip after alloBMT. The impact on clinically relevant endpoints such as the subsequent incidence of fractures and avascular necrosis in these patients will determine the utility of this intervention.
The Grey Zone between Burkitt and Diffuse Large B Cell Lymphomas: Impact of Chromosomal Breakponts at Myc and Other Loci.
Authors: Haralambieva Eugenia , Boerma Evert-jan , van Imhoff Gustaaf , Rosati Stefano , Schuuring Ed , Mueller-Hermelink Hans-Konrad , Kluin Philip M. , Ott German
Published: October 2019

A prompt distinction of Burkitt lymphoma (BL) versus diffuse large B cell lymphoma (DLBCL) has important clinical implications. We analyzed 74 adult grey zone lymphomas (BL/DLBCL) and 10 reference pediatric BL using immunohistochemistry for Ki-67, CD10, bcl2 and bcl6, and Fluorescence In Situ Hybridization (FISH) for MYC, BCL2 and BCL6 breakpoints. Four hematopathologists reached a final (consensus) diagnosis independently in 80% of the reference BL, 24% of the test BL and 69% of the DLBCL. A MYC breakpoint was detected in all reference BL, 95% of the test BL and 35% of the DLBCL. BCL2 and BCL6 breakpoints were infrequent in BL (5 and 6%) and DLBCL (13% and 16%). Three BL and 5 DLBCL contained double breakpoints. A phenotypic shift to BL in the DLBCL group was indicated by the expression of CD10 and bcl6 in 67% and 91% of the cases, respectively. One third of all lymphomas showed a classical genotype and expression pattern of BL (MYC breakpoint+, Ki-67>90%, CD10+, bcl6+, bcl2-) but only 63% of these cases were classified as BL. Our data indicate that a subgroup of DLBCL shares markers with BL, which is probably due to an overlapping histogenesis of both tumors.

Value of immunohistochemistry and additional FISH for BCL2 and BCL6 breakpoints to reach the consensus diagnosis in 38 MYC breakpoint carrying cases of adult grey zone BL / DLBCL lymphomas

BCL2 and BCL6 breakpoint no restriction not present no restriction not present 38 / 74 cases carried a MYC/8q24 breakpoint phenotype no restriction no restriction Ki67>90%, CD10+, bcl6+, bcl2- Ki67>90%, CD10+, bcl6+, bcl2- N 38 30 24 22 Burkitt (%) 20 (53) 17 (57) 15 (63) 15 (68) DLBCL (%) 18 (47) 13 (43) 9 (37) 7 (32)