bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific MiRNA Silencing by Lentivirus Mediated Antagomir Expression in Chronic Myeloid Leukemia (CML) Cells.
Authors: Scherr Michaela , Venturini Letizia , Battmer Karin , Schaller-Schoenitz Michael , Schaefer Daniel , Dallmann Iris , Ganser Arnold , Eder Matthias
Published: October 2019

Micro RNAs (miRNA) are small non-coding RNAs that regulate gene expression by specific hybridization to complementary sequences in the 3′ untranslated region of corresponding mRNAs. Concomitant recruitment of specific multi-protein complexes results either in inhibition of mRNA translation or mRNA degradation. miRNAs are processed in a regulated multi-step process from primary transcripts into mature miRNAs by cellular components which are also at least partially involved in the process of RNA interference (RNAi). Aberrant expression of specific miRNAs has recently been described in human lymphoma and leukemia. In particular, BCR-ABL and c-MYC dependent over-expression of the polycistronic and oncogenic miR-17-92 cluster (encoding miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92) has been described in chronic myeloid leukemia (CML) cell lines, primary CD34+ cells from CML patients (Venturini et al. 2007), and in lung cancer. In BCR-ABL positive K562 cells, miR-17-92 encoded miRNAs repress luciferase activity in miRNA-specific reporter assays. In addition, lentivirus-mediated over-expression of miR-17-92 increases both cell proliferation and sensitivity to imatinib induced cell death. To analyse the function of individual miRNAs of the miR-17-92 polycistron, we generated lentivirus-based strategies to induce stable miRNA-specific loss- and gain-of function phenotypes for miR-18a, miR-19b, and miR-20a, respectively. Over-expression of miRNAs embedded within miR-30-derived sequences from an internal SFFV-LTR promoter allows isolation of K562 cells with increased miRNA expression. In contrast, expression of complementary oligonucleotides (antagomirs) from a H1 promoter located in the lentiviral 3′LTR can induce stable hypomorphic miRNA-phenotypes. In lentivirally transduced K562 cells, individual silencing of miR-18a, miR-19b, and miR-20a by the corresponding antagomirs (ant-miR-18a, ant-miR-19b, ant-miR-20a) specifically relieves miRNA-mediated reporter gene repression. Correspondingly, inhibition of miRNA-function correlates to reduced ‘miRNA’-amplification by miRNA-specific quantitative RT-PCR. Furthermore, protein expression of E2F-1, a known miR-20 target, is enhanced by lentivirally expressed anti-miR-20 in a dose-dependent manner, whereas over-expression of miR-20a reduces E2F-1 levels. Finally, combined over-expression of specific miRNAs and antagomirs reveals specific induction of cell proliferation by miR-18a but strong inhibition by miR-20a in K562 cells, respectively. In contrast, anti-miR-18a, but not anti-miR-19b, anti-miR-20a, or control antagomirs inhibits proliferation of K562 cells. These data demonstrate individual and complementary functions of miR-17-92 encoded miRNAs in CML and identify potential targets for specific therapeutic intervention on the miRNA level.
Cbl-b Deficiency Enhances Motility and Impairs Leukemogenesis by Bcr-Abl.
Authors: Badger-Brown Karla M. , Bailey Monica L. , Penninger Josef , Barber Dwayne L.
Published: October 2019

One of the prominent tyrosine phosphorylated substrates of BCR-ABL identified in cells from chronic-phase Chronic Myeloid Leukemia (CML) patients is the Cbl ubiquitin ligase and adaptor protein. Bone marrow transplantation studies have revealed that Cbl is dispensable for CML development, as animals receiving Cbl-deficient bone marrow cells expressing BCR-ABL develop a myeloproliferative disease (MPD) with a similar latency and phenotype as wild type bone marrow recipients (Dinulescu et al. 22: 8852–60, 2003). Cbl belongs to a family of E3 ubiquitin ligases that also contains Cbl-b and Cbl-3. In contrast to the equivalent Cbl protein expression displayed in hematopoietic cell lines expressing BCR-ABL, addition of the oncogene leads to a decreased expression of the related family member, Cbl-b. The objective of this study was to determine whether Cbl-b is a critical signaling intermediate downstream of BCR-ABL. To investigate this objective, we performed retroviral transduction of wild type- and Cbl-b- deficient bone marrow with BCR-ABL and utilized these cells for bone marrow transplantation of lethally irradiated mice. Recipients of wild type donor cells transduced with the p210 isoform of BCR-ABL, succumb to a rapidly fatal CML-like MPD, while the lack of Cbl-b produces a significant delay in morbidity (average latency of 32 days as compared with 18 days for recipients of wild-type marrow). Expansion of differentiated neutrophils, as visualized by histopathological analysis, is evident in the peripheral blood, bone marrow and spleen of all transplanted animals. However, circulating white blood cells are augmented in number (average wbc count for Cbl-b(+/+), 150 × 106 cells / mL; Cbl-b(−/−), 83 × 106 cells / mL) and enhanced spleen size is observed in Cbl-b(+/+) mice (35 mg / g of body mass) as compared to Cbl-b(−/−) (19 mg/g) animals. Liver masses were comparable between the two transplants. In depth flow cytometric studies of splenic cells reveals the characteristic decrease in B and T cells and enhanced myeloid lineages. Notably, an increased number of granulocytes (Gr-1+/Mac-1+ cells) is observed in the Cbl-b-knockout animals, suggesting enhanced migration but decreased survival of leukemic cells in the Cbl-b−/− spleen. In support of this hypothesis, fibroblasts deficient in Cbl-b display enhanced chemotactic migration toward Fetal Calf Serum. As migration is a key determinant of cellular retention in the bone marrow, the loss of Cbl-b could ultimately be affecting cell localization. Therefore, our data supports a continued study of Cbl-b as a motility regulator in BCR-ABL-dependent CML progression.
Growth Factor Independent 1b (Gfi1b) Is Highly Expressed in Human CML and Accelerates p210BCR-ABL Induced Leukemia in Mice.
Authors: Vassen Lothar , Tenen Daniel G. , van der Reijden Bert A. , Duehrsen Ulrich , Moroy Tarik , Mahlmann Stefan , Ebeling Peter , Jansen Joop H.
Published: October 2019

Chronic myeloid leukemia (CML) is one of the most frequent leukemic diseases and invariably associated with a reciprocal t(9;22) translocation which creates a juxtaposition of the BCR and ABL genes to form the p230-, p190- or p210BCR-ABL constitutively active tyrosine kinases. This constitutive tyrosine kinase activity is the cause of CML and other leukemic diseases. CML is very efficiently treated with small molecule tyrosine kinase inhibitors, but CML is a stem cell initiated disease and surprisingly the leukemic stem cells cannot be eradicated by tyrosine kinase inhibitor treatment, resulting in a high risk of the development of therapy resistance. To design more successful therapies, it is primordial to understand the molecular mechanisms underlying this disease. Here we show that growth factor independent 1b (Gfi1b), also translocated in CML, is strongly induced in peripheral blood mononuclear cells (PBMCs) in patients with chronic- or acute myeloid leukemia (AML) as well as in patients suffering from myeloprolifarative syndrome (MPS) or B-lymphoblastic leukemia (B-ALL). Gfi1b is a transcriptional repressor essential for erythroid- and megacaryocytic cells, but also expressed in hematopoietic stem cells, early myeloid precursors and mature myeloid cells in peripheral blood. Interestingly, the expression of Gfi1b is further induced by long term treatment with tyrosine kinase inhibitors like Glivec, although the patients are phenotypically in remission. Additionally we describe a new splice variant of Gfi1b which is overrepresented in CML, AML and B-ALL, but not in MPS. Using a mouse model system we demonstrate, that Gfi1b strongly accelerates p210BCR-ABL induced leukemogenesis. We further show that Gfi1b inhibits the activity of PU.1, a key molecule in hematopoiesis and repressor of leukemogenesis. Our findings bear considerable significance with regard to the role of Gfi1b as an oncogene in human leukemia and to its possible value as a new target for leukemia therapy.
Long-Term Efficacy of Dasatinib in Chronic-Phase CML: Results from the Phase I Trial (CA180002).
Authors: Cortes Jorge , Sawyers Charles L. , Kantarjian Hagop M. , Shah Neil P. , Paquette Ronald , Paliwal Prashni , Bleickardt Eric , Talpaz Moshe
Published: October 2019

Imatinib resistance in CML is frequently associated with BCR-ABL mutations. Such kinase domain mutations are emerging as a common mechanistic theme in cases of acquired resistance to kinase inhibitor therapy. Five-year follow-up from the IRIS trial indicates that the incidence of hematologic resistance to imatinib is 17% and >30% of patients have discontinued imatinib therapy on study. Preclinical studies reveal that dasatinib (SPRYCEL®), a novel BCR-ABL inhibitor, is 325-fold more potent than imatinib in inhibiting the kinase activity of BCR-ABL, as well as demonstrating activity against all imatinib-resistant BCR-ABL mutations tested to date, with the exception of T315I. Here we present an update of a Phase-I dose-escalation study, with enrollment between November 2003 and April 2005, of dasatinib in imatinib-resistant or -intolerant patients with CML or Ph+ ALL; this report focuses on the long-term follow-up of the 45 patients (56% male) with late chronic-phase disease. Dasatinib was administered at doses of 15–180 mg/day on a QD or BID schedule. Median duration of CML prior to entry was 8 years (range 1–17). Median age was 63 years (range 28–79). Prior therapy included interferon-α in 91% of patients and stem-cell transplantation in 4%; 62% had received prior imatinib doses >600 mg. With a minimum follow-up of 27 months, the rate of complete hematologic response (CHR) was 91% (41/45). Major cytogenetic responses (MCyR) were attained in 51% of patients (23/45), with complete CyR (CCyR) in 44% (20/45). The rates of CCyR for the QD and BID schedules were 45% (10/22) and 43% (10/23), respectively. The median duration of CHR and MCyR have not been reached with a median duration of treatment of 28 months. In a landmark analysis, the 36-month progression-free survival (PFS) rate was 87% and overall survival (OS) was 94% for patients who achieved a MCyR within the first year of dasatinib therapy. For patients who did not achieve a MCyR within the first year, the 36-month PFS figure was 28% and OS was 68%. These long-term data confirm the durability and depth of hematologic and cytogenetic response associated with dasatinib in patients who have failed treatment with imatinib. Evidence is also provided to substantiate that the significant response rates observed with dasatinib therapy in this population translate into prolonged progression-free and overall survival. Dasatinib therefore has the potential to positively impact on the natural history of CML.
Imatinib Dose Can Be Safely Reduced after Complete Cytogenetic Response (CCyR) in Patients (pts) with Chronic Myeloid Leukemia (CML) in Early Chronic Phase (CP) Treated with High-Dose Imatinib.
Authors: Jain Nitin , Kantarjian Hagop M. , Fava Carmen , Thomas Deborah , Burger Jan A. , Borthakur Gautam , Pate Odeal , Cortes Jorge
Published: October 2019

Background Imatinib (standard dose, SD, 400mg/d) is standard therapy in CP CML. Several observations suggest that initial therapy with high-dose (HD) imatinib might be more effective. We previously reported that HD imatinib (800mg/d) leads to significantly better rates of complete cytogenetic response (CCyR), major molecular response (MMR) and complete molecular response (CMR), and improved event-free survival compared to SD. However, HD imatinib is associated with more frequent grade 3–4 hematological toxicity compared to SD. In addition, 36% pts required dose reduction due to toxicities. We investigated the outcome of pts who were maintained on 800mg/d compared to those whose dose was reduced from 800mg/d.

Methods Pts enrolled in 3 sequential trials using imatinib for frontline therapy for CP CML were analyzed: 208 treated with HD and 50 with SD. Primary end points were event-free survival (EFS) and transformation-free survival (TFS).

Results The median age was 48 years (range 17–84 years). Median follow up was 49 months. 4 year event-free survival (EFS) and transformation-free survival (TFS) were significantly better for pts treated with HD imatinib compared to SD (EFS: 92% vs. 77%, p=0.01; TFS: 98% vs. 89%, p=0.0045). Among pts treated with HD, 100 (48%) had a dose reduction at any time [600mg/d (n=50), 400mg/d (n=38), 300mg/d (n=12)]. Of them, 45 (45%) were dose-reduced after achieving CCyR including 26 (26%) after MMR. The median time to dose reduction was 3 months (range 1–60 months). For pts starting with HD imatinib, there was no difference between pts continuing with HD and those with a dose reduction in terms of EFS at 4 years (92% in both groups, p=0.65) and TFS at 4 years (98% vs. 97%, respectively, p=0.36). However, EFS at 4 years was significantly worse if the dose reduction was done prior to achievement of CCyR (84% vs. 100%, p=0.02). Similarly, patients who were dose-reduced before MMR had significantly worse 4 year EFS (88% vs. 100%, p=0.02). EFS curve for patients who had dose reduction done prior to CCyR was similar to that of pts treated with SD imatinib (p=0.59).

Conclusions These results indicate that pts treated with HD imatinib can be safely dose reduced once they have achieved CCyR (preferably MMR) without having any impact on EFS or TFS. Such dose reduction may help decrease side effects as well as cost of the treatment. A prospective evaluation of this approach is warranted.
Toll-Like Receptor 2 and 4 Are Expressed on Transplanted T-Cell Subsets and Influence Outcome after Allogeneic Unrelated Peripheral Blood Stem Cell Transplantation.
Authors: Platzbecker Uwe , Stoehlmacher Jan , Goekkurt Eray , Pabst Caroline , Thiede Christian , Ehninger Gerhard , Bornhäuser Martin
Published: October 2019

Introduction: Recently, Toll-like receptor (TLR) 2 and 4 have been identified as the most important receptors for LPS, which is contained in the cell wall of gram-negative bacteria and is known to be a main inducer of graft versus host disease (GVHD). The role of TLR expressing T-cells within the graft for the induction of GVHD in patients after unrelated peripheral blood stem cell transplantation (PBSCT) is unknown.

Methods and patients: We therefore determined by flow cytometry expression of TLR 2 and 4 on T-cells within the graft of 63 patients receiving unrelated PBSCT after intensive conditioning followed by cyclosporine A and methotrexate as GVHD prophylaxis. Additionally, donor specific single nucleotide polymorphisms (SNP) for TLR2 (R753Q), TLR4 (D299G) and TLR4 (Y135A) were determined. The data were finally correlated with clinical endpoints.

Results: As expected, TLRs were not expressed on T-cells in peripheral blood of healthy donors (TLR 2: <1.0%, TLR4 <0.5% of T-cells, n=6). In contrast we detected a distinct up-regulation of these receptors on T-cells within the grafts. TLR2 and TLR4 expression on CD4+ T-cells ranged from 1.2%–12.3% (median 3.1%) for TLR2 and from 1.2%–12.0% (median 3.7%) for TLR4. Among the CD8+ T-cells 0.9%–16.1% (median 3.3%) expressed TLR2 and 0.7%–13.3% (median 3.5%) expressed TLR4. The SNP for TLR2 (R753Q) and TLR4 (D299G) was found in 8.6% and 10.3% of the allogeneic donors, respectively but did neither correlate with the expression levels of TLR on T-cells nor with clinical endpoints. Treatment-related mortality from infections was observed in 10 patients (16%). Interestingly, higher expression of TLR2 and 4 on CD4+ but not CD8+ T-cells was significantly associated with an increased cumulative incidence of fatal infections (38% vs. 8% p=0.03 for TLR2 and 37% vs. 9% p=0.007 for TLR4). Neither the overall CD3+, CD4+ and CD8+ cell dose nor the expression of TLR2 and 4 on CD4+ and CD8+ T-cells showed a significant association with the incidence of acute or chronic GVHD or relapse.

Conclusion: These data suggest a previously unrecognised up-regulation of TLR, on CD4+ and CD8+ T-cells contained in G-CSF mobilized apheresis products. Whether these phenotypic changes impact on T cell function or patient outcome warrants further investigation.
Acute Graft-Versus-Host Disease Following Umbilical Cord Blood Transplantation: Retrospective Survey Involving 2015 Japanese Patients.
Authors: Miyakoshi Shigesaburo , Yamaguchi Takuhiro , Kami Masahiro , Matsumura Tomoko , Yuji Koichiro , Narimatsu Hiroto , Kusumi Eiji , Sakamaki Hisashi , Kouzai Yasushi , Okada Masaya , Osugi Yuko , Kobayashi Ryuji , Inoue Masami , Takahashi Satoshi , Murata Makoto , Kai Shunro , Kato Koji , Nagamura Tokiko , Taniguchi Shuichi , Kato Shunichi K.
Published: October 2019

BACKGROUND Limited information is available on incidences and clinical features of acute graft-versus host disease (GVHD) after cord blood transplantation and large-sized researches have been awaited.

METHODS We investigated the incidences and clinical features of acute GVHD in 2,015 patients reported to the Japan Cord Blood Bank Network, who underwent cord blood transplantation between June 1997 and August 2006.

RESULTS Of 2,015 patients, 1481 patients (73%) achieved neutrophil engraftment at a median of day 22 (range, 6–81). Cumulative incidence of neutrophil recovery at day 100 was 0.74 (95%CI, 0.73–0.76). Of 2015 patients, 708 patients developed grade II-IV acute GVHD: grade II (n=423), grade III (n=237), and grade IV (n=48). The median onset was day 19 (range, 4–190). The cumulative incidences of grade II-IV and III-IV acute GVHD at day 100 were 0.35 (95% CI, 0.33–0.37) and 0.14 (95% CI, 0.12–0.15), respectively. Skin and gastrointestinal acute GVHD was documented in 1,006 and 405 patients, respectively, whereas liver GVHD was diagnosed in 149 patients. Multivariate analysis identified the following predictors of grade II-IV acute GVHD: the number of infused nucleated cells, transplantation from female donors to female recipients, TBI-containing preparative regimens, methotrexate-containing GVHD prophylaxis, and tacrolimus-based GVHD prophylaxis. Overall survival rates at three years of patients with grade 0-I, II and III-IV GVHD who survived 100 days or longer were 0.58 (95%CI, 0.53–0.63), 0.61 (95% CI, 0.54–0.67) and 0.40 (95%CI, 0.31–0.49), respectively.

CONCLUSIONS Acute GVHD following cord blood transplantation is mild and has graft-versus malignancy effects.

Probability of event free survival after cord blood tranplantation in the patients with grade 0-I, II and III-IV who survived 100 days or longer Event - free survival of the patients with grade 0-I, II and III-IV GVHD who survived 100 days or longer was 0.54 (95% CI, 0.49–0.59), 0.58 (95%CI, 0.52–0.65) and 0.41 (95%CI, 0.32–0.49),respectively,3 years after transplantation. Probability of event free survival after cord blood tranplantation in the patients with grade 0-I, II and III-IV who survived 100 days or longer Event - free survival of the patients with grade 0-I, II and III-IV GVHD who survived 100 days or longer was 0.54 (95% CI, 0.49–0.59), 0.58 (95%CI, 0.52–0.65) and 0.41 (95%CI, 0.32–0.49),respectively,3 years after transplantation.
Is Splenectomy Necessary in Patients with Myelofibrosis and Extensive Splenomegaly Prior to Allogeneic Stem Cell Transplantation with Reduced Intensity Conditioning Regimens?.
Authors: Ciurea Stefan O. , Sadegi Barry , Wilbur Andrew , Alagiozian-Angelova Victoria , Gaitonde Sujata , Dobogai Lisa C. , Akard Luke P. , Hoffman Ronald , Rondelli Damiano
Published: October 2019

Allogeneic stem cell transplantation has been shown to restore normal hematopoiesis in patients with intermediate or high risk primary myelofibrosis (PMF) or myelofibrosis preceeded by polycythemia vera or essential thrombocythemia. However, in patients with PMF and extensive splenomegaly it is not clear whether transplant is associated with an unacceptable risk or if splenectomy should be performed prior to transplant. In this study, ten consecutive patients with myelofibrosis who were not splenectomized received an allogeneic hematopoietic stem cell transplantation (HSCT) from related or unrelated donors and were periodically monitored by ultrasound or CT scan for 12 months after transplant to assess the kinetics of the reduction of splenomegaly. These findings were correlated with the time to resolution of marrow fibrosis, time to engraftment and clinical outcome. Over a 12 month period a progressive reduction in spleen size was observed in all the patients and paralleled the reduction in marrow fibrosis (Figure 1). Of 10 patients, 5 with a splenic longitudinal diameter >30 cm showed a more prolonged time to engraftment ANC> 0.5 × 109/L (d 19±5 vs 13±2, p=0.05) and platelet > 20 × 109/L (d 75±104 vs 11±2, p=0.06). However, of the 5 patients with more extensive splenomegaly only 2 experienced delayed engraftment of platelets (d 77 and 256, respectively). Full donor chimerism was observed in all patients within 60 days after transplantation regardless of the size of the spleen. At a median follow-up of 54 months (range: 2–80) all the patients are alive but 1 who died of a TTP like syndrome 2 months after transplant and had a small spleen at the time of transplant. A clinical CR has been documented in 7 patients and 2 have achieved clinical improvement as assessed by IWG response criteria. These findings suggest that allogeneic HSCT with RIC regimen can be safely performed in patients with extensive splenomegaly. Although in these patients the time to engraftment may be prolonged, the significant risk of morbidity and mortality associated with splenectomy may be avoided.

Figure Figure
The Importance of Harmonizing Transplantation Procedures in Children with ALL: ALL-SCT-BFM 2003, a Prospective Multicenter Trial.
Authors: Schrauder André , Stackelberg Arend , Schrappe Martin , Klingebiel Thomas , Holter Wolfgang , Sykora Karl-Walter , Handgretinger Rupert , Ebell Wolfram , Pötschger Ulrike , Zimmermann Martin , Karlhuber Susanne , Peters Christina
Published: October 2019

As results of frontline and relapse protocols for children with acute lymphoblastic leukaemia (ALL) are improving over time, there is a strong need for prospective haematopietic stem cell transplantation (SCT) trials, ensuring well standardized procedures for all relevant components which are potentially responsible for heterogeneity in post-SCT outcome. Therefore, in 2003 the Berlin-Frankfurt-Münster (BFM)-Study Group initiated a prospective international multicenter trial (ALL-SCT-BFM 2003). Main goals are the standardization and harmonisation of the SCT procedure and the question, if nowadays SCT from a matched sibling donor (MSD) is equivalent to SCT from a very well matched donor (MD). Further, we want to determine the efficacy of a SCT from HLA-mismatched donors (MMD) compared to SCT from MD/MSD as well as the incidence of acute and chronic graft versus host disease (GvHD) after SCT. Between September 2003 and June 2007, 381 patients (185 in CR1, 196 in CR2 or after subsequent relapse) were recruited by 27 participating SCT centers in Austria, Germany and Switzerland. Indications for SCT were poor response to induction treatment, either detected by morphology on day 33 or by minimal residual disease load measured after 11 weeks of treatment, cytogenetic aberrations [t(9;22), t(4;11)], early bone marrow relapses of ALL, or any subsequent ALL relapse. 76 patients had an indication for a MSD, 131 patients for a MD, and 174 patients for a MMD. Compliance with HLA-typing procedures according to the trial guidelines (high resolution typing of HLA A, B, C, DRB1 and DQB1) improved over time: 54% in 2004, 83% in 2005, 90% in 2006, and so far 100% in 2007. Unmanipulated bone marrow was used in 73% of MSD- and MD-SCT as recommended by the protocol. The recommended conditioning regimens (TBI+VP16 for MSD, TBI+VP16+ATG for MD, and TBI+Flu+VP16+ATG for MMD; in children younger than 2 years, TBI was substituted by BU+Cy) were used in 80% of all patients. GvHD prophylaxis (cyclosporine A for MSD, and cyclosporine A plus “short methotrexate” for MD) was given in 83% of all patients according to the trial guidelines. In most cases, deviations were due to clinical reasons. As the trial ALL-SCT-BFM 2003 is ongoing till 2009, interim results regarding outcome will not be presented. Nevertheless, treatment-related mortality is still very low (7%) and has improved over time since the very early beginning. Final results will be a very stable basis for subsequent ALL-SCT trials, focussing on controlled modifications and interventions in those patients being at highest risk for relapse subsequent to SCT.
The Transfusion Iron Overload Score Is a Potential Predictor of Survival in Stem Cell Transplant Patients.
Authors: Storey Jonathan A. , Connor Rebecca , Lewis Zachary T. , Lovato James , Hurd David , Torti Frank M. , Molnar Istvan
Published: October 2019

Iron overload at the time of bone marrow transplantation has recently been associated with increased mortality. The mechanism by which iron overload negatively impacts survival is unclear. In an effort to better define iron overload and its potential role in the transplant setting, we examined 78 consecutive patients at risk for transfusion-related iron overload (AML, ALL, MDS, and aplastic anemia) undergoing either autologous or allogeneic stem cell transplant at our institution between 1999 and 2004. To estimate transfusion-related iron overload, we devised a novel scoring system based on routinely available laboratory variables obtained prior to transplant. In our scoring system, a single point was awarded for each of the following:

number of red cell units transfused prior to transplantation > 25; serum ferritin > 1000 ng/ml; and a semi-quantitative bone marrow iron stain of 6+.

The Transfusion Iron Overload Score (range of 0 to 3) predicted for overall survival in our patient sample (Fig. 1, p = 0.0003 by Kaplan-Meier log rank trend) and was more powerful than using serum ferritin alone (p = 0.02). The calculated score retained its significant association with overall survival while controlling for traditional risk factors (age, sex, diagnosis, transplant type, donor relation, and remission status), inflammation (c-reactive protein), and measures of organ damage (transaminases, total bilirubin, INR, and cardiac ejection fraction) (p = 0.04 by Cox proportional-hazards regression). The 27 patients (35%) with a high score (≥ 2) had a dramatically lower median survival of 5.0 months compared to 29.3 months in those with an iron score of less than two (unadjusted hazard ratio of 2.5; 95% CI of 1.6 to 6.3). The observed mortality seen in those with a high score was primarily attributable to treatment-related mortality (p = 0.018) rather than disease-related mortality (p = 0.48). This finding likely accounts for the significant survival impact of iron overload seen in the allogeneic (p = 0.006) but not the autologous (p = 0.48) subgroup. In conclusion, the Transfusion Iron Overload Score served as a strong independent predictor of survival in our stem cell transplant population, identifying patients at significant risk for early treatment-related mortality. Our results lend further support to the notion that iron overload is detrimental in the transplant setting, and for the first time suggest that transfusion-related iron overload increases mortality via a mechanism independent of pre-existing inflammation and end-organ dysfunction.

Figure 1. Overall post-transplant survival by the Transfusion Iron Overload Score. Figure 1. Overall post-transplant survival by the Transfusion Iron Overload Score.
Prognostic Impact of Elevated Pretransplant Serum Ferritin in Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Mahindra Anuj , Bolwell Brian , Sobecks Ronald , Rybicki Lisa , Brown Stacey , Pohlman Brad , Dean Robert , Andresen Steve , Kalaycio Matt , Sweetenham John , Copelan Edward
Published: October 2019

Iron overload is a well-established adverse prognostic factor in patients who undergo hematopoietic stem-cell transplantation (HSCT) for thalassemia and appears to play a similar role in patients with acute leukemia and myelodysplastic syndrome. We report the results of a large study of consecutive patients undergoing myeloablative allogeneic transplantation to assess the influence of pretransplantation ferritin on treatment-related mortality (TRM), graft-versus-host disease (GVHD) and survival. Pretransplantation ferritin, obtained within 100 days preceding transplant, was available on 222 consecutive patients ≥18 years of age undergoing allogeneic HSCT from 10/20/2000 to 12/04/2006. Transplantation was performed for various disorders (AML n= 105, ALL= 44, CML= 25, MDS= 19, NHL=18, and other= 11) and mainly with busulfan-based preparation (n=153). One hundred twenty four donors were siblings and 98 unrelated. The median patient age was 43 years. Median ferritin level was 1433 ng/ml (range 7–17029). Seventy-five patients had ferritin levels >1910 ng/ml. Median follow-up for surviving patients was 2.5 years. Recursive partitioning analysis identified significantly lower survival for patients with serum ferritin >1910 ng/ml as shown: ESTIMATED SURVIVAL BY PRETRANSPLANTATION FERRITIN LEVEL

Figure Figure

Age, gender, disease, disease status at transplant, interval from diagnosis to transplant, number of prior chemotherapy regimens, prior radiation therapy, ferritin, albumin, AST, Alkaline phosphatase, LDH, preparative regimen and CD 34+ dose elevated were assessed. In a multivariable model for survival, elevated ferritin was a significant,independent,adverse prognostic factor for survival (p=0.011, HR=1.68). The addition of albumin, a negative acute phase reactant, did not change the prognostic impact of elevated ferritin. Patients with ferritin >1910 ng/ml had a higher incidence of TRM(p=0.046), as illustrated below: INCIDENCE OF TRM BY FERRITIN LEVELS

Figure Figure

This siginificantly higher incidence of TRM occured despite a significantly lower incidence of chronic GVHD (p=0.019) and a trend towards a lower incidence of acute GVHD (p=0.10) in patients with elevated ferritin levels. The incidences of hepatic venoocclusive disease and relapse mortality were not different between the two groups.

Conclusions: Elevated ferritin is an independent adverse risk factor in a large group of patients undergoing myeloablative transplantation for various diseases. Higher TRM occured despite the association of elevated ferritin with a significantly decreased incidence of chronic GVHD, consistent with an immunosuppressive effect demonstrated in other settings. Trials should be undertaken to analyze the benefit of iron chelation therapy prior to BMT in patients with iron overload.
SAGE Analysis Demonstrates Increased Expression of TOSO Contributing to Fas Mediated Resistance in CLL.
Authors: Proto-Siqueira Rodrigo , Panepucci Rodrigo A. , Careta Francisco P. , Rizzatti Edgar G. , Lee Abigail , Clear Andrew , Morris Kelly , Falcão Roberto P. , Zago Marco A. , Gribben John G.
Published: October 2019

The molecular pathogenesis of CLL remains unclear; therefore we sought to identify new genes involved in the pathogenesis or mechanisms of resistance in CLL and other B cell malignancies. To this end we performed SAGE in CLL cells and compared results with those from healthy B cells (nCD19+). Gene expression profiles were obtained by SAGE and individual gene expression was measured by real time PCR. Analysis revealed only 27 genes with significant (p< 0.001) differences between mutated and unmutated subtypes of CLL cells. In contrast to the high level of similarity between the CLL subtypes, comparison of the complete CLL SAGE libraries versus nCD19, revealed that 56 genes were over represented and 50 genes were down regulated in CLL (fold change higher than 20x, p<0.001 as a cut-off). A gene ontology analysis using the top 1,000 transcripts revealed some genes aberrantly expressed in CLL not previously reported. One of these is TOSO (regulator of Fas-induced apoptosis) of interest since CLL cells are resistant to Fas mediated killing. Real-time RT-PCR analysis of 78 cases of CLL and 12 nCD19+ confirmed that TOSO mRNA was over expressed (4.4 fold) in CLL (p<0.0001), and its expression was positively correlated with BCL-2 expression. There was no correlation between TOSO expression and prognostic factors, including IgVH mutational status, cytogenetics, evolution or previous treatment. In addition, using tissue microarray of 1mm cores arranged in triplicate followed by indirect immunohistochemistry we evaluated TOSO protein distribution and intensity in 44 CLL lymph nodes and 5 reactive tonsils and appendix. Scoring was performed by two histopathologists independently and demonstrated that more than 50% of CLL cases express TOSO strongly (2+, 3+) with a range of 10% to 95% of positive tumour cells (p=0.0057, Chi-Square test), whereas among control samples analyzed all were negative except the mantle cells from one tonsil and one appendix were scored as weakly positive (1+); the remainder were negative. Flow cytometry analysis in 12 CLL cases and 5 healthy subjects demonstrated a 5.6 fold increase of TOSO in peripheral CLL cells (p=0.013). SAGE results confirmed that both CLL subtypes share a similar expression profile and highlighted a novel gene, TOSO. This protein plays a functional role upstream of Fas in the extrinsic apoptosis pathway and ongoing studies are assessing the mechanisms whereby TOSO mediates resistance of CLL cells to anti-Fas treatment and ways to reverse this.
Mutual Regulatory Loop between miR-155 and PU.1 Is a Candidate Pathogenesis Factor in CLL.
Authors: Stopka Tomas , Vargova Karin , Kokavec Juraj , Pospisil Vit , Curik Nikola , Burda Pavel , Skoultchi Arthur I. , Zavadil Jiri
Published: October 2019

MicroRNAs are non-coding post-transcriptional regulators of gene expression often dysregulated in human cancers including hematological malignancies. MiR-155 is a putative oncogenic factor found overexpressed in chronic lymphocytic leukemia (CLL) cells. We have confirmed the increased levels of miR-155 in peripheral blood lymphocytes in 12 patients with chronic lymphocytic leukemia (CLL) compared to 11 normal controls, using quantitative PCR assay. Among the predicted high-scoring hematopoietic target genes of miR-155 is the ets-family transcription factor PU.1, a key regulator of myelo-lymphoid lineage development. Using gene expression profiling of CLL peripheral lymphocyte samples compared to normal controls, we have determined significant downregulation of the PU.1 mRNA and of additional numerous miR-155 targets previously reported to be required for myelo-lymphoid differentiation, and of a set of gene targets of PU.1-chromatin DNA interactions that we determined by chromatin immunoprecipitation analyses. Functional pathway analysis of the miR-155 targets downregulated in CLL patients suggest the suppression of T-cell receptor pathway, TGF-b/Bmp, MAPK, Jak/Stat and Wnt signaling and remodeling of actin cytoskeleton and cell-matrix interactions, as well as the regulation of numerous transcription regulators including c-FOS, c-MYC, MAFB and HDAC4. Recent reports suggested a role of miR-155 in lymphocytic differentiation, we have tested whether PU.1 may regulate expression of its own regulator miR-155. We used two cell systems expressing a transgene encoding PU.1 fused with the ligand binding domain of the estrogen receptor, ER):

murine erythroleukemia (MEL) cells (Rao 1997) and PU.1 null hematopoietic progenitor cells stably transfected with PU.1-ER (Walsh 2002).

Using a miRNA microarray assay we have identified a set of 21 miRNAs that are modulated by PU.1. Indeed, miR-155 was among the upregulated targets alongside a known PU.1 miR target miR-233. The direct interaction between miR-155 promoter and PU.1 protein was confirmed by chromatin immunoprecipitation in MEL cells. In summary, our data suggests that increased miR-155 levels may contribute to CLL by inhibiting PU.1 function and suppressing PU.1-mediated programs and that in two independent conditional PU.1 cell systems PU.1 directly binds miR-155 promoter and upregulates its expression suggesting thus a mutual regulatory mechanism between PU.1 and miR-155.
Blood Induced Cartilage Changes in a Hemophilia A Murine Model.
Authors: Dunn Amy L. , Shehata Bahig M.
Published: October 2019

Background: Hemophilia A results from a congenital deficiency of clotting factor VIII. Patients with hemophila are unable to generate adequate thrombin during blood coagulation and therefore have a bleeding diathesis. Degenerative joint disease is the largest source of morbidity for hemophilic patients. The pathophysiology of this process is poorly understood but clinically, repeated bleeding into a joint leads to changes in the synovium, articular cartilage and underlying bone. It is not well known how quickly cartilage changes occur or if animal age has any impact on susceptibility to blood induced cartilage damage.

Materials and Methods: The E16 fVIII knockout mouse is a well-established model for hemophilia research. These mice contain a targeted disruption of the factor VIII gene at exon 16 (E16) in a C57BL/6 background. Despite having no detectable circulating fVIII activity they rarely suffer from spontaneous hemarthroses. Therefore hemarthroses was mimicked by injecting 5 μL of autologous whole blood into the hind knee. Six male mice per group in each of three ages; 12, 24 and 52 weeks were utilized. The animals were sacrificed 48 hours after injection. The injected and control contralateral knee joint from each animal was fixed in 10% formalin and then mounted in paraffin. After decalcification, five micrometer sections through the joint were obtained. Joint architecture was examined after hematoxylin and eosin staining. Proteoglycan content of articular cartilage was evaluated after alcian blue staining as part of the modified Mankin score. The pathologic specimens were also scored utilizing Valentino’s visual bleeding score. The Student’s t-test was utilized for significance testing.

Results: All mice showed evidence of blood remaining in the injected joint at 48 hours. The mean visual bleeding score was significantly higher for the injected knees versus the control knees of all age groups p<0.001. The mean visual bleeding score was significantly between the 12 and 24 week animals p=0.0112 and the 12 and 52 week animals p=0.0379, but not between the 24 and 52 week animals p=0.5423. The modified Mankin score was significantly higher between the injected and control knees of all age groups as seen in Figure 1 where the mean and standard deviation is shown for 6 animals in each group. Interestingly, one control knee in a 24 week old animal demonstrated synovial hyperplasia and decreased proteoglycan staining but had no evidence of blood in the joint. This likely represented a previous spontaneous hemorrhage. The modified Mankin score was highest for the 12 week old animals and lowest for the 52 week animals but did not achieve statistical significance p=0.5746.

Conclusions: Injection of autologous blood mimicked clinical hemarthrosis. Evidence of decreased proteoglycan staining of articular cartilage was evident within 48 hours of blood exposure. Additionally, there was a trend toward more prominent proteoglycan loss in the youngest animals.

Figure Figure
Phase I Trial of HuLuc63 in Multiple Myeloma.
Authors: Bensinger William , Zonder Jeffrey , Singhal Seema , Mohrbacher Ann , Dean Robert M. , van Rhee Frits , Al-Homsi Ahmad-Samer , Munshi Nikhil C. , Conner Edward R. , Hussein Mohamad A.
Published: October 2019

Background: HuLuc63 is a humanized monoclonal antibody directed against CS1 that has demonstrated potent in vivo and in vitro activity against myeloma cells in pre-clinical models. CS1 is a cell surface glycoprotein expressed at high levels on myeloma cells, plasma cells and to a lesser extent on a subset of CD8 + lymphocytes and natural killer (NK) cells. A Phase I study was initiated to evaluate the maximum tolerated dose (MTD) of HuLuc63 in a population of relapsed refractory multiple myeloma patients who have been exposed to at least two prior therapies. In a traditional phase 1 design, six potential dosing cohorts are planned with 3 to 6 patients enrolled per cohort to evaluate safety, pharmacokinetics (PK), biologic activity and clinical response. Each course of HuLuc63 is given IV at the same dose every 2 weeks for a total of 4 doses at a level of 0.5, 1, 2.5, 5, 10, 20 mg/kg. An additional 4 doses may be administered if the patient has demonstrated at least stable disease during the initial course of therapy. To date, a total of 7 patients have been treated at 2 dose levels; 3 in the 0.5 mg/kg cohort and 4 in the 1.0 mg/kg cohort. Median patient age is 64 years and the median number of prior therapies is five. An additional patient was enrolled in the 1.0 mg/kg cohort as a patient in that cohort had a 2-week delay in receiving his second dose due to worsening pain at the site of a pre-existing lytic bone lesion. No dose-limiting toxicities have occurred. Infusions were well tolerated, with grade 1 and 2 toxicities occurring during or after the first dose including chills (2 patients), flushing, pyrexia, rigors, dyspnea and fatigue. Patients who reported first dose reactions were pretreated with acetaminophen and diphenhydramine prior to subsequent doses and only one patient reported fatigue after the second dose. Four serious adverse events have occurred in 2 patients which were considered unrelated to HuLuc63. These include migraine headache (twice in the same patient), congestive heart failure and hypercalcemia. Two patients discontinued therapy prematurely due to progressive disease but all other patients have received 4 doses of HuLuc63. No clinical responses have been observed. In xenograft mouse models, a consistent drug level of 10 mcg / mL was required to show minimal biologic activity. Preliminary PK data reveals that peak serum drug levels for the 0.5 mg/kg dosing cohort reached 10 mcg / mL, which was sufficient to achieve CS 1 saturation of at least 70% on the antigen rich NK cell subset. Drug levels dropped below 1 mcg/mL by day 7, however, coinciding with a decrease in saturation. This indicates that the higher doses to be used in subsequent cohorts may achieve and surpass sustained concentrations in patients above this level. Thus far, HuLuc63 appears to be well tolerated in patients with multiple myeloma. Enrollment is continuing to determine the MTD.
In-Situ Proliferation May Explain the Persistence of Host Derived Langerhans Cells Following Allogeneic Hematopoietic Stem Cell Transplantation with Reduced Intensity Conditioning.
Authors: Richards Julie , Pearce Laurence , Patten Piers E.M. , Buggins Andrea G.S. , Ho Aloyisius Y. , Pagliuca Anthony , Mufti Ghulam J. , Devereux Stephen
Published: October 2019

Langerhans cells (LCs) are antigen presenting cells found in the epidermis. They are thought to originate from bone marrow precursors although studies in mice suggest they have the ability to self renew in situ. Murine models have also shown that recipient LCs are required for the development of acute graft versus host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-HSCT). Engraftment of LCs following human allo-HSCT has previously been assessed using migration from isolated epidermal sheets, which may be inaccurate because of the potential for differential migration of donor and recipient cells. We therefore studied LC engraftment using fluorescence immunophenotyping and simultaneous in-situ hybridisation for X/Y chromosomes in sex mismatched transplants. Skin biopsies were performed at days 28, 56 and 100, 6 months and 1 year post transplant on 8 patients receiving alemtuzumab based RIC allo-HSCT regimens. Ten micron cryosections were taken, LCs labelled with anti-CD1a and X/Y chromosomes detected with the CEP X/Y probe kit. Slides were examined on a Zeiss LSM510 Meta confocal microscope and Z-stack images were collected to cover a volume of 100μm3. Chimerism of purified CD15+ and CD3+ peripheral blood cells was determined in parallel by polymerase chain reaction amplification of short tandem repeat sequences. Results are tabulated below and show that compared to CD15+ myeloid blood cells, donor LC engraftment is markedly delayed (day 28, 56 and 100 paired t-tests give P<0.0001, P=0.001 and P=0.0302 respectively). Of the 8 patients studied, 1 went on to develop biopsy proven GVHD. In all cases studied, recipient LCs could still be detected albeit in small numbers at the 1-year time point. Since these findings suggested that epidermal LCs are either very persistent or replicate in-situ, we next stained samples of normal and post transplant GVHD skin with both CD1a and the proliferation marker Ki67. In both cases a proportion of CD1a+ cells also expressed Ki67 (13.5% of normals, n=8; 18.6% of GVHD n=9 p=0.33). Both recipient and donor derived LCs appear to be capable of proliferating since both male and female Ki67+ CD1a+ cells were identified in 2 cases for which appropriate samples were available. In conclusion, our results provide independent confirmation that recipient LCs may persist for a prolonged period following allo-HSCT and show that at least in part, this may be due to self renewal within the epidermis.

Post transplant chimerism analysis Cell Type Day 28 Day 56 Day 100 6 Months 1 Year Median % donor chimerism at specific time points following allo-HSCT. LC 18.5 (n=8) 73 (n=7) 90 (n=7) 93.5 (n=6) 97 (n=3) CD3+ 99 (n=8) 80.5 (n=6) 66.5 (n=6) 79.5 (n=4) 100 (n=3) CD15+ 100 (n=8) 100 (n=6) 100 (n=6) 100 (n=4) 100 (n=2)
GMP-Conform Generation and Cultivation of Unrestricted Somatic Stems Cells (USSC) from Cord Blood Using the SEPAX©-Separation Method a Closed Culture System Applying Cell Stacks.
Authors: Radke Teja F. , Buchheiser Anja , Lefort Aurélie , Maleki Mahtab , Wernet Peter , Kögler Gesine
Published: October 2019

Generation and characterization of unrestricted somatic stem cells (USSC) from cord blood (CB) was described by our group and has been well established under laboratory conditions [Koegler et al 2004, 2005 and 2006; Sensken et al 2007]. Due to their proliferative and differentiation capacity, USSCs are an interesting candidate for the future development of cellular therapy for tissue repair and tissue regeneration as well as a supportive cell layer to support hematopoietic reconstitution. Since generation and expansion under GMP-grade conditions is mandatory for use in clinical application, the automated cell processing system Sepax (BIOSAFE) with the CS900 separation kit was used for mononuclear cell separation and the subsequent generation of the USSC colonies in the presence of 30% GMP-grade fetal calf serum (Perbio), low-glucose DMEM-medium/10-7M dexamethasone. Expansion of USSC was performed in a closed system (Macopharma) applying cell stacks (Costar Corning). Results achieved so far indicate that the generation frequency and quality of generated USSC under GMP conditions are equal or even superior (45%) to manual generation under laboratory conditions (43%). 20 cord-blood units (mean volume 88,5 +− 15,8 ml; mean number of mononuclear cells 3,1 +−0,6 *108 MNC) have been processed, resulting in 9 USSC-colony formations and lines within 14–28 days. Growth kinetics is equal to the previously established USSC-lines (∼36–48 h / population doubling). Analysis of the immunophenotype as well as the differentiation potential towards the mesenchymal, neural and endodermal lineages also showed no difference to those lines generated manually using Ficoll-separation and normal cell culture flasks (Costar Corning T225). The closed system applied here is perfectly suitable to ensure safe and easy handling of the USSC, including seeding, trypsination and harvesting. In combination with the cell stack system (1, 2, 5 and 10 layers), cell amounts of more than 1.0×109 USSC can be achieved within 4 passages. These USSC products were temperature controlled cryopreserved in the presence of 10% DMSO, HSA and dextran. USSC can be thawed and further expanded in clinical grade quality. On the basis of their pluripotency and expansion under GMP-conditions into large quantities, these USSC from cord blood, when pretested for infectious agents and matched for the major transplantation antigens, may serve as a universal allogeneic stem cell source for tissue repair and tissue regeneration.