bluuebunny/crossref_metadata_2025 huggingface dataset:
Multiple Integration Events into Several Putative Oncogenes Was Required To Cause Leukemogenesis in Two Primate Recipients of RCR Contaminated Stem-Cells.
Authors: Pule Martin A. , Rousseau Alexandra , Kang Elizabeth , Ueda Takahiro , von Kalle Christof , Nienhuis Arthur W. , Heslop Helen , Vanin Elio , Tisdale John F.
Published: October 2019

Integrating vectors are essential for stem-cell and T cell gene-therapy approaches to enable passage of the transgene to subsequent progeny, and retroviral vectors have been the commonly utilized for clinical applications. However, insertional events can potentially cause malignant transformation of transduced cells. In 1991, adult rhesus macaques received CD34+ cells transduced with a NEO expressing retroviral vector which was inadvertently contaminated with ampho-pseudotyped replication competent retrovirus(RCR). Three primates (88053, 15445 and 88049) developed a rapidly progressive lymphoid neoplasm 6–7 months after transplantation characterized by diffuse lymphadenompathy, hepatosplenomegaly and a large thymic mass. Immunohistochemistry of the neoplasm revealed a CD8+ CD2+ alphabeta TCR+ phenotype strongly positive for RCR. The neoplasm was preceeded by increasing amphotropic envelope detectable in peripheral blood by PCR which was ultimately detectable in 100% of peripheral blood leukocytes. Tissue is available for study from primates 15445 and 88049. Genomic Southern blotting revealed multiple integrants, more in 15445 than in 88049. Using a simplified technique (FLEA-PCR) for generating libraries of Integrant-Host-Junctions (IHJ), we studied leukemia samples from these two (primates 15545 and 88049). So far we have characterized 52 different flanking sequences from primate 15545 of which we could match 43 genomic loci. We have isolated 39 different flanking sequences from primate 88049 of which we could match 29 genomic loci. Of the characterized integration sites from 15445 the following potential oncogenes could be affected: DKK3, CUTL1, RUNX1, RUNX3, TCF7L2, FOXP1, MTIF2, GAB1, CST7, CABIN1 and RHOH. Of the characterized integration sites from 88049 the following potential oncogenes could be affected: LY86, JARID2, TNIK(TRAF2 and NCK interacting kinase), SMT3H2, PTK2B, MSF, FBXL10 and RAPH1. Both primate leukemia samples contain an integration site just 5 to the gene for Neuronal acetylcholine receptor protein (CHRNA9). RNA derived from leukemic bone marrow from both animals and involved thymic tissue from one was subjected to microarray analysis and compared to that of control tissue using the Affymetrix human genome U133 plus 2 array. The gene expression profile was similar among diseased tissues, and efforts to correlate the expression profile with genes potentially dysregulated by integration events is ongoing. Together with some subjects of the recent X-SCID study, these macaques represent the only cases of leukemogenesis caused by vector induced insertional mutagenesis in primates. Our findings confirm the assumption that with an inert transgene (NEO, RCR), multiple integrations effecting different oncogenes are required to result in malignant transformation of transduced cells.
Gene Transfer to Repopulating Human CD34+ Cells Using Amphotropic, GALV or RD114 Pseudotyped HIV-1 Based Vectors from Stable Producer Cells.
Authors: Richter Johan , Johansson Maria , Relander Thomas , Olsson Karin , Ikeda Yasuhiro , Takeuchi Yasuhiro , Collins Mary
Published: October 2019

A novel, stable human immunodeficiency virus type 1 (HIV-1) vector packaging system, STAR, was tested for its ability to transduce human cord blood CD34+ progenitor cells assayed both in vitro and after transplantation to NOD/SCID mice. Vectors pseudotyped with three different gammaretrovirus envelopes were used; the amphotropic MLV envelope (MLV-A), a modified gibbon ape leukemia virus envelope (GALV+) and a modified feline endogenous virus RD114 envelope (RDpro). Titration of vectors harvested from the stable packaging cells gave the following results: MLV-A: 1–3x106 inf.u/ml, GALV+: 1x106 inf.u/ml and RDpro: 3–10x106 inf.u/ml CB CD34+ cells were transduced on Retronectin® preloaded with vector either in fresh medium (PL alone) or in vector containing medium (PL+VCM). Gene transfer to freshly thawed CD34+ in the absence of cytokines was very low. Addition of cytokines (CK; SCF, TPO and FL) increased gene transfer efficiency significantly and this was further augmented if the cells were prestimulated for 24 hours wit the same cytokines. Concentration of the vectors (15-fold) by low-speed ultracentrifugation increased gene transfer to CD34+ cells in vitro even further. More than 90% of cells were transduced with a single exposure to the RDpro vector as determined by GFP expression using flow cytometry. The two other pseudotypes transduced approximately 70% of the cells under the same conditions. Detailed in vitro transduction data is shown in table as percentage GFP positive cells:

RDpro MLV-A GALV+ PL alone PL+VCM PL alone PL+VCM PL alone PL+VCM No CK 9.2±2 0.9±0.8 12.5±3.5 6.6±0.1 0.9±0.4 1.4±0.5 With CK 22.4±8.3 3.3±5.3 18.2±3.8 10.8±3.8 4.8±1.7 16.7±9.0 CK prestim f 24 hours 57.6±14.9 35.3±32.1 45.4±14.9 36.3±14.8 3.4±1.7 19.5±12.2 Concentrated vector CK prestim f 24 h 93.4±3.3 93.3±3.7 73.3±7.6 40.3±20.4 66.1±7.5 77.8±10.9

Transplantation of CD34+ cells prestimulated for 24 hours and then transduced with a single hit of concentrated vector to irradiated NOD/SCID mice revealed that the RDpro vector transduced 55.1% of NOD/SCID repopulating human cells which was significantly higher than the MLV-A (12.6%) or GALV+ (25.1%) pseudotyped vectors. Thus, the STAR packaging system, especially with the modified RD114 envelope, appears to be a new valuable tool for the genetic modification of primitive human progenitor cells.
The Human Xist Gene Promoter Prevents Silencing of an Integrated Reporter Gene.
Authors: Greene Michael R. , Lowrey Christopher H.
Published: October 2019

Epigenetic silencing and position dependent expression are long-standing problems which continue to limit the development of gene replacement therapy. As a strategy to overcome this problem we have tested the ability of the human XIST (X inactivation-specific transcript) gene promoter to overcome epigenetic silencing. The XIST gene is one of a relatively small number of genes which are expressed from the inactive X chromosome. The product of this gene is an untranslated structural RNA which coats the X chromosome destined for inactivation prior to H3 Lys 9 hypoacetylation, H3 Lys 9 methylation, CpG island methylation and the subsequent silencing of most of the genes on the chromosome. Continued expression of the XIST gene in this highly repressive environment is required to maintain the chromosome in an inactive state. The region of the proximal promoter of the XIST gene on the inactive X chromosome has been shown to retain an active chromatin structure. Based on these findings we hypothesized that the XIST gene promoter would be able to resist the epigenetic changes which lead to transgene silencing. To test this idea, we subcloned a minimal XIST promoter upstream of an enhanced GFP reporter gene in a pUC-based plasmid which also contained a neomycin resistance gene. The same plasmid, with a CMV promoter, served as a control vector. The plasmids were electroporated into mouse erythroleukemia (MEL) cells and then grown in media containing G418. The MEL cell line was chosen because genes transferred into these cells are frequently silenced and because it is often used as a first screen for vectors with potential for use in therapeutic gene transfer to erythroid cells. Individual colonies were selected and G418 removed. After expansion of the clones, flow cytometry was used to determine the percentage of cells in each clonal population which were expressing GFP as determined by comparison to the untransduced MEL cell line. Silencing typically involves a gradual decrease in the proportion of cells expressing the integrated transgene. Statistical analyses of results were performed using the t-test. 16 XIST and 13 CMV clones were available for analysis at the start of the experiment (time 0). 11/13 CMV clones and 12/16 XIST clones initially expressed GFP. Of the clones which were expressing GFP, the average percentages of positive cells were higher for those with the XIST promoter (63% vs. 41%, p= 0.015). Expression was reanalyzed after 6 weeks of culture in the absence of G418 selection. At this time point, the average percentage of GFP expressing cells was much higher for the XIST clones (57% vs. 19%, p=0.00008) and when analyzed for silencing, XIST clones were expressing at an average of 90% of their time 0 levels vs. 46% for the CMV clones (p=.0008). These results indicate that the XIST promoter is resistant to silencing in our model system and is a candidate for further development and mechanistic studies.
Clinical Relevance of Recipient Leukocyte Infusion (RLI) Therapy.
Authors: Saito Toshiki I. , Rubio Marie T. , Sykes Megan
Published: October 2019

BACKGROUND

Surprisingly, anti-tumor responses can occur in patients who reject donor grafts following nonmyeloablative hematopoietic cell transplantation (Dey et al., Biol Blood Marrow Transplant 7:604). In murine mixed chimeras prepared with nonmyeloablative conditioning, we previously showed that recipient leukocyte infusions (RLI) induced anti-tumor responses against host-type tumors (Rubio et al. Blood 102:2300).

To further investigate the clinical relevance of this RLI model, we:

Evaluated the effect of RLI from tumor-bearing mice Compared RLI with allogeneic lymphocyte infusion in untreated mice

METHODS

Mixed chimerism was achieved in BALB/c (H-2d) mice conditioned with depleting anti-CD4 and CD8 mAbs on Day-5, cyclophosphamide 200 mg/kg on Day -1 and 7 Gy thymic irradiation on Day 0 prior to transplantation of 25x106 B10.BR (H-2k) bone marrow cells. Some groups received RLI (3x107 BALB/c spleen cells) seven weeks post-BMT. Some RLI donor mice received BALB/c A20 B cell lymphoma cells (1x105) two weeks before RLI. Some groups received RLI depleted of B cells using B220 negative selection by MACS column. A20 cells (5x105) were given i.v. one week after RLI in chimeras or after allogeneic lymphocyte infusion (3x107 B10.BR spleen cells) to untreated BALB/c mice.

RESULTS

In the clinical setting, RLI would be obtained from tumor-bearing hosts. We therefore examined whether RLI is still effective when the lymphocytes are obtained from the tumor-bearing mice. Contamination of B220+ cells in the RLI was less than 0.03%. RLI recipients from the tumor-bearing mice (n=10) showed similar tumor survival compared to recipients of RLI from naïve donors (n=10) (median survival time [MST] 53 versus 50 days respectively, p=0.48) (Figure 1). Thus, with a purging procedure, the same anti-tumor effect was achieved with RLI from tumor-bearing hosts as from non-tumor-bearing hosts.

Allogeneic lymphocyte injection is a potentially feasible anti-tumor therapy. We therefore compared anti-tumor effects of allogeneic lymphocyte infusion into naïve mice with that of RLI given to mixed chimeras. Chimeric RLI recipients mice (n=13) had longer survival (MST 61 days) than naïve mice receiving allogeneic lymphocytes (n=10) (MST 39.5 days, P<0.01) (Figure 2). This result suggests not only that the anti-tumor effect of RLI therapy is stronger than allogeneic lymphocyte infusion therapy but also suggests that rejection of allogeneic cells is insufficient and mixed chimerism is required prior to the induced rejection to achieve maximum anti-tumor effects.

CONCLUSION

Together, these data reinforce the potential of RLI therapy to be a new HSCT strategy which does not have the risk of graft-versus-host disease.
Cord Blood CD4+CD25+ Derived T Regulatory Cell Lines Express FoxP3 Protein and Manifest Potent Suppressor Function.
Authors: Godfrey Wayne R. , Spoden Darrin J. , Ge Ying G. , Baker Seth R. , Liu Baoling , Levine Bruce L. , June Carl H. , Blazar Bruce R. , Porter Stephen B.
Published: October 2019

CD4+CD25+ T regulatory (Treg) cells have been shown to critically regulate self and more recently allograft tolerance in mice. Studies of human Treg have been hindered by the presence of CD25-dim conventional T cells that copurify during Treg isolation. We compared adult and cord blood sources for Treg, with the hypothesis that cord blood should lack significant numbers of memory cells or environmentally reactive T cells. We found that cord blood was a superior source for Treg isolation compared to adult blood. CD4+CD25+ cells were readily purified, and generated cell lines that consistently exhibited potent suppressor activity, with >95% suppression of allogeneic MLR (29/30 donors). The cells could markedly suppress an allo-MLR at a 1/16-1/32 suppressor/responder cell ratio. The cultured Treg cells blocked cytokine accumulation in MLR, with a less robust inhibition of chemokine production.

Cultured Treg uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular CTLA4. Upon re-stimulation with anti-CD3/CD28 beads, the cultured Treg produced minimal cytokines (IL2, IFN-gamma, and IL10), and preferentially expressed TGF-beta latency associated protein on the cell surface, while conventional CD4+CD25− derived cell lines did not. Cytokine production however, could be largely restored by stimulation with PMA/ionomycin. Abundant FoxP3 mRNA was detected in fresh, cultured, and anti-CD3/CD28 bead restimulated CD4+CD25+ cells (approximately 2–4 fold less than cyclophillin A). Low amounts of FoxP3 mRNA were present in fresh CD25− cells, and they increased 32 fold on culture. However, FoxP3 protein, as assessed by western blot, was specifically expressed in the CD25+ derived cell lines, and was not detected in the CD25− derived cell lines. Restimulation with anti-CD3/CD28 beads led to increased expression of FoxP3 protein in CD25+ derived cell lines, but not in CD25− derived cell lines.

Cord blood derived cultured suppressor cells were not cytolytic in chromium release assays, and mediated suppression of alloreactivity that was cell contact dependent and predominantly independent of IL10 and TGF-beta. Antibodies to GITR, GITR-L, OX40, OX40-L, CTLA4, and PD1 did not significantly affect suppression of the culture activated Treg cells. These results demonstrate virtually pure populations of potent suppressor cells can be cultured from cord blood, and these cell lines form an ideal model system for the evaluation of suppressor cell biology and mechanisms of action.
Identification of Oncogenic Collaborators Providing Insights into the Tumor Suppressing Function of the Eed Polycomb Group (PcG) Gene.
Authors: Sauvageau Martin , Post-Doc Julie Lessard , Miller Michelle , Sauvageau Guy
Published: October 2019

Polycomb Group (PcG) proteins are key regulators of normal and leukemic hematopoiesis (Lessard and Sauvageau, Nature 2003; Lessard and al., Genes Dev 1999). The eed gene is part of a PcG complex together with Ezh2 and Suz12. Evidence from our laboratory indicates that eed plays a crucial role in the negative regulation of the proliferative capacity of lymphoid and myeloid progenitors, suggesting that this PcG gene is a tumor suppressor. Interestingly, the human eed locus is in a region of chromosome 11 (11q14.2-22.3) which is associated with recurrent deletions in several reported cases of B-CLL, mantle cell lymphoma (MCL) and T-PLL. We now show that when exposed to ionizing radiation at 5 weeks of age, both eedhypo/hypo and eednull/+ mutant mice developed mono- or oligoclonal B and T cell lymphomas with much shorter latencies than observed in littermate controls (50% lethality at 16 wks; 27 wks and 34 wks post-irradiation for eedhypo/hypo, eednull/+ and controls, respectively). We reasoned that new pathways involved in tumorigenesis could be at play in this eed sensitized background, potentially allowing for the identification of new oncogenic collaborators. Therefore, a proviral insertional mutagenesis screen was conducted. Neonatal MMLV infection of eed mutant mice dramatically accelerated the occurrence of B and T cell lymphomas (50% lethality at 16 wks; 22 wks and 26 wks post-infection for eedhypo/hypo,eedhypo/+ and controls, respectively). To characterize retroviral insertions inverse-PCR (IPCR) was performed using genomic DNA isolated from 32 eedhypo/hypo and 27 wild-type tumors. More than 780 IPCR products were recovered and purified. To date, at least 140 retroviral insertion sites (RIS) have been cloned, sequenced and mapped against the mouse genome. Of these, 7 insertion targeting gene loci known to be cancer-related, like Pim1 (n= 7 tumors), Notch1 (n= 14 tumors) and c-Myc (n= 3 tumors) have been identified. In addition, 8 new common insertion sites (CIS), such as Socs1 (n= 6 tumors), Gadd45g (n= 5 tumors) and Fgfr3 (n= 6 tumors) have also been identified. Rearrangements of the Pim1 locus were found in more than 22% of the eed1989/1989 mutant mice whereas none could be found in wild-type littermates. Moreover, RT-PCR analysis indicates that the Pim1 gene is clearly overexpressed in 6 out of 16 eed1989/1989 tumors analyzed compared to wild-type, further supporting a key role for the Pim1 pathway in eed sensitized tumor cells. Thus, the data presented in this study provide evidence that eed has tumor suppressing function. It also points to a novel mechanism of tumor suppression by a PcG protein. Based on these results, it will be interesting to study the status of eed and of the newly identified CISs in human diseases associated with deletions of 11q14.2-22.3.
Low-Dose Administration of Recombinant IL-2 Following T Cell Depleted Bone Marrow Transplantaion Induces Expansion of CD4+CD25+ FOXP3+ Regulatory T Cells in Vivo.
Authors: Zorn Emmanuel , Lista Despina , Kim Haesook , Bellucci Roberto , Canning Christine , Soiffer Robert J. , Ritz Jerome
Published: October 2019

Regulatory T cells (Treg) play a key role in controlling immune responses following allogeneic hematopoietic stem cell transplantation (HSCT). In murine models, infusion of purified CD4+CD25+ Treg at the time of transplant is sufficient to prevent acute GVHD. In humans, development of acute as well as chronic GVHD has been associated with reduced numbers of Treg following allogeneic HSCT, suggesting that defective reconstitution of this functional cell type can contribute to exacerbation of alloimmune responses. Based on these results, adoptive cellular therapy using purified and in vitro expanded populations of Treg has been proposed as a therapeutic strategy to correct chronic GVHD. Treg are mainly characterized by the constitutive expression of the IL-2 receptor ? chain, CD25 and proliferate in response to IL-2 in vitro. In vivo, the effects of IL-2 on Treg populations are unknown. To examine this question we quantified changes in Treg in 9 patients with CML who previously received low dose IL-2 following allogeneic HSCT. Patients enrolled in this protocol received a daily intravenous infusion of 2 X 105 U IL-2/m2 for 3 months, starting 3 months after CD6 depleted allogeneic bone marrow transplantation (BMT). No patient developed GVHD following IL-2 administration and overall toxicity was minimal. The predominant immunologic effect of IL-2 reported in the initial study was a marked increase in NK cell populations characterized as CD3-CD16+CD56+ as well as total CD56+ cells. In this retrospective analysis we investigated populations of CD4+CD25+ T cells before and 1 to 2 months after the beginning IL-2 treatment. Using RNA extracted from patient PBMC we also assessed the level of expression of the specific transcription factor FOXP3 by quantitative PCR as an alternate marker of Treg in vivo. As initially reported, all 9 patients showed a marked increase in CD3-CD56+ cells 1 to 2 months post IL-2 administration. In contrast, the percent of CD3+ T cells were either unchanged or slightly decreased. The percent of CD4+CD25+ cells within the CD3+ T cell population increased during IL-2 treatment (median: 5.8 pre IL-2 vs 7.6 post IL-2, p-value=0.02). Likewise, FOXP3 expression in the CD3+ population showed 5 to 19 fold increase in 8 of 9 patients during this period (median: 3817 AU pre IL-2 vs. 18924 AU post IL-2, p-value=0.055). These results indicate that administration of low dose IL-2 can augment Treg cells in vivo as reflected by increased ratio of CD4+CD25+/CD3+ T cells as well as higher levels of FOXP3 expression. These studies suggest that prolonged treatment with low dose IL-2 can effectively expand CD4+CD25+ Treg in vivo. This represents a novel strategy for expanding regulatory T cells in vivo and may be useful alone or in conjunction with adoptive cellular therapy with Treg.
The Synthetic Pentasaccharide, Fondaparinux, Reduces Inflammation and Neutrophil Accumulation in Kidney Ischemia-Reperfusion Injury.
Authors: Schabbauer Gernot , Frank Rolf D. , Holscher Todd , Sato Yuichiro , Tencati Michael , Pawlinski Rafal , Mackman Nigel
Published: October 2019

Acute inflammatory diseases are often accompanied by coagulation activation leading to local thrombotic complications and disseminated intravascular coagulation. Recent studies support the concept of crosstalk between coagulation and inflammation. The synthetic pentasaccharide, fondaparinux, is a selective antithrombin-dependent inhibitor of coagulation factor Xa. In this study, we investigated the effect of fondaparinux in a lethal murine model of kidney ischemia-reperfusion (I/R) injury that is associated with coagulation and inflammation. Fondaparinux treatment of I/R-injured mice significantly reduced serum creatinine levels and increased survival from 0 to 44% compared with saline treated control mice. In contrast, depletion of fibrinogen with ancrod was not protective, suggesting that fondaparinux may have additional properties beyond its anticoagulant activity. Indeed, fondaparinux significantly reduced IL-6 and MIP-2 expression but did not reduce MCP-1 expression. Furthermore, fondaparinux significantly decreased neutrophil accumulation in the injured kidneys. Finally, we showed that fondaparinux reduced recruitment of neutrophils into the peritoneum in a model of acute peritonitis and inhibited the binding of U937 cells to P-selectin in vitro. Our data indicate that fondaparinux has both anticoagulant and anti-inflammatory activity reducing fibrin deposition and blocking the binding of inflammatory cells to activated endothelium. Fondaparinux may be useful in the treatment of acute inflammatory diseases.
TNFα Mediated Facilitating Cell Enhancement of Hematopoietic Stem Cell Function in Vivo and In Vitro Involves Bcl-3.
Authors: Rezzoug Francine , Huang Yiming , Tanner Michael K. , Wysoczynski Marcin , Shannie Carrie L. , Ratajczak Mariusz Z. , Fugie-Vivier Isabelle J. , Ildstad Suzanne T.
Published: October 2019

Approaches to enhance engraftment of hematopoietic stem cells (HSC) are of primary interest in BM transplantation. CD8+TCR− facilitating cells (FC) improve HSC engraftment in allogeneic recipients without causing graft versus host disease. FC also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients, suggesting that FC act directly on HSC. We therefore analyzed the mechanism for FC-mediated effects on HSC. We found that FC increased the ability of purified HSC to generate colonies in both the CFC and CAFC/LTC-IC assays, confirming a direct effect of FC on different HSC compartments. Co-incubation of HSC with FC for 18 or 40 hours significantly increased the survival of HSC and their subsequent ability to generate CFC at these same time points. We determined that the anti-apoptotic effect of FC on HSC was associated with the up regulation of anti-apoptotic Bcl-3 transcripts. We postulated here that the effect of FC on HSC was due to cytokine secretion. As FC produce TNFα after CpG ODN stimulation and TNFα has various activities on HSC, we evaluated the role of TNFα on FC function. FC were sorted from TNFα deficient mice and the facilitative activity of FC on HSC engraftment was assessed. FC from TNFα deficient mice were impaired in facilitating HSC engraftment in both the syngeneic and allogeneic models, suggesting a role for TNFα in FC function. Notably, TNFα transcripts were present in FC by 16 hours of co-incubation of FC + HSC and FC produce TNFα (surface and intra-cellular) when in contact with HSC. Furthermore, when TNFα was blocked (using anti-TNFα mAb), FC from wild type mice lost the ability to increase HSC clonogenicity (from 38.2±13.6 CFC/1000 HSC in HSC alone to 65.7 ± 22 for HSC + FC and 38.2 ± 19.6 for FC pre-incubated 1 hour with anti-TNFα mAb before incubation with HSC). Moreover, anti-TNFα mAb also blocked the ability of FC to up-regulate Bcl-3 transcripts in HSC. In conclusion, FC act directly on HSC via several mechanisms to maintain the balance between proliferation/differentiation/survival of HSC. One central mechanism implicates TNFα production by FC, which may protect HSC from undergoing apoptosis by up-regulating anti-apoptotic transcripts (e.g. Bcl-3). These findings may have great impact for the use of accessory cells in HSC transplantation, especially when numbers of HSC are limiting.
Gi Protein Signals Are Required for BM Homing of Hemopoietic Progenitor Cells, and Cooperate with Alpha4-Intergrin and Endothelial Selectins.
Authors: Bonig Halvard , Priestley Greg V. , Jiang Yi , Papayannopoulou Thalia
Published: October 2019

Several examples suggest a relationship between in vitro migratory capacity and bone marrow (BM) homing. Pertussis toxin (PTX) is a potent inhibitor of Gi protein coupled receptor signaling and as such, it blocks hemopoietic progenitor cell (HPC) migration in vitro. However, contrary to expectation, no effects on BM homing were observed in previous studies. We therefore re-examined the effect of PTX on BM homing of HPC. We found that prolonged incubation of BM cells or fetal liver cells with PTX inhibited their BM homing in irradiated or non-irradiated recipients by >75%. A concomitant increase in circulating CFU-C in blood provided added assurance that the data were not due to non-specific toxicity. This inhibition of BM homing was of functional consequence, since PTX-treated cells provided impaired radioprotection and markedly decreased short-term engraftment. Detailed studies showed that inhibition of in vitro migration and ribosylation of Gi proteins were complete only after extended incubation with PTX, whereas short-term incubation for 1–2 h, as used in previous studies, was insufficient. In addition, the incubation of BM cells with SCF may have exaggerated the negative effect of PTX on the inhibition on BM homing. We next sought to test the basis of the residual 25% homing of PTX treated cells. As PTX did not directly inhibit hemopoietic cell adhesion, we tested homing of PTX-incubated and control BM cells in genetic models deficient in certain adhesion molecules on either the hemopoietic cells or the hemopoietic microenvironment. BM homing of PTX-treated α4integrin-/- BM cells transplanted into wild-type (WT) mice, or PTX-treated WT cells transplanted into EPselectin-/- recipients inhibited BM homing significantly stronger than either modality alone, to >90% and >95%, respectively. Of note, untreated WT cells have normal homing in EPselectin-/- mice. These data demonstrate cooperativity between Gi blockade and α4integrin or endothelial selectin blockade in inhibiting BM homing of progenitor cells. Absence of β2integrin or L-selectin in the presence or absence of PTX did not inhibit BM homing. In summary, these studies show that Gi protein signals are required for BM homing of progenitor cells. PTX is a very strong inhibitor of BM homing, suggesting that it may inhibit convergent signals from more than one mediator. We further show that the residual BM homing of migration-impaired BM cells is due to participation of α4 integrin on the transplanted cells, or to endothelial selectins on the host side.
Involvement of PKCe in the Negative Regulation of Akt Activation Stimulated by G-CSF.
Authors: Liu Hong , Dong Fan
Published: October 2019

Granulocyte colony-stimulating factor (G-CSF) supports the proliferation, differentiation and survival of myeloid cells by stimulating the activation of several signaling cascades including the serine/threonine kinase Akt pathway. Akt activation has been shown to be important for G-CSF-induced survival and granulocytic differentiation. Although significant progresses have been made in our understanding of the molecular mechanisms by which Akt is activated, much less is known about the signaling events that negatively regulate Akt activation. Interestingly, G-CSF-induced activation of Akt was completely inhibited when myeloid 32D cells transfected with the wild type G-CSF receptor were incubated with phorbol-12-myristate 13-acetate (PMA), a PKC activator. PMA-mediated inhibition of Akt activation occurred with 5 min and lasted at least 1 hour. Previously, it has been shown that a carboxyl terminally truncated G-CSF receptor (D715), whose expression is associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia (SCN), mediates significantly prolonged Akt activation. Notably, Akt activation by G-CSF in 32D cells expressing the D715 receptor mutant was rapidly downregulated by PMA treatment. The inhibitory effect of PMA on Akt activation was abolished by pretreatment of cells with the specific PKC inhibitor GF109203X, suggesting that PKC-dependent pathway negatively regulates Akt activation. Ro-31-7549, a specific inhibitor of PKCe, also abrogated PMA-mediated inhibition of Akt activation whereas rottlerin and Go6976, inhibitors of PKCd and PKC a/bI,, respectively, displayed no effect. Together, these results identified PKCe as being critically involved in PMA-mediated inhibition of Akt activation. Experiments are currently under way to determine the mechanism by which PKCe downregulates Akt activation and the role of PKCe in the regulation of cell proliferation, differentiation and survival in response to G-CSF.
Thrombocytopenia Is More Common and Nadir Occurs Earlier with Unfractionated Heparin Than with Low Molecular Weight Heparin - Results from the Prospective Catch Registry.
Authors: Moll Stephan , Abrams Charles S. , Rice Lawrence , Becker Richard C. , Berger Peter B. , Anstrom Kevin J. , Honeycutt Emily F. , Crespo Eric M. , Granger Chris , Moliterno David J. , Rodgers Jo E. , Steinhubl Steven R. , Tapson Victor F. , Ohman Magnus
Published: October 2019

Background : Thrombocytopenia in the patient on heparin can have various etiologies, including benign reversible causes and serious, potentially life-threatening ones, such as heparin induced thrombocytopenia (HIT). Knowledge about the prevalence of and timecourse of thrombocytopenia in heparin-treated patients may be helpful to develop systems of alerting clinicians to possible HIT and determining how frequently to check blood counts to detect thrombocytopenia that may be heralding HIT.

Methods : The CATCH registry is a prospective registry of inpatients enrolled between March 2003 and April 2004 at over 50 US hospitals in 3 strata: [1] receiving > 96 hours of unfractionated heparin (UFH) or low molecular weight heparin (LMWH), [2] developing thrombocytopenia (platelets >50% reduction from baseline or <150,000/mm3) in the cardiac care unit and [3] patients on whom HIT tests were ordered. Here we present the data of the prolonged heparin stratum.

Results : 1,121 and 861 patients received UFH and LMWH, respectively, for > 96 hours. A platelet count decrease of > 50 % from baseline was seen more frequently in patients on UFH than in patients on LMWH (10.7% and 7.9 %, respectively; p = 0.03). The parameters that predicted development of thrombocytopenia in the UHF group were length of heparin therapy, body mass index, and admission to a cardiac service; the only parameter predicting thrombocytopenia in the LMWH group was length of LMWH treatment. Of the patients with decreased platelet count by > 50 % from baseline (for UFH n = 120; for LMWH n = 68), this drop occurred in the UFH group at a median of 3.0 days after initiation of heparin and at a median of 4.0 days in the LMWH group. The difference in timing between the 2 groups was not statistically significant (p = 0.205). The platelet nadir was reached after a median /mean of 4.0 / 7.4 days in the UFH group, and 8.0 / 11.4 days in the LMWH group. This was statistically significantly different between the 2 groups (p-value = 0.0025).

Conclusions : A platelet count decrease of > 50 % from baseline occurs frequently in inpatients treated with prolonged heparin. It occurs slightly more frequently on UFH than on LMWH. The median time to onset of thrombocytopenia (> 50 % decrease from baseline) occurs early, at 3–4 days. Daily platelet count checks in the first few days of heparin therapy may be helpful to rapidly discover thrombocytopenia that may then prompt HIT testing.
The VEINES-QOL/Sym Questionnaire Is a Valid and Reliable Measure of Quality of Life in Patients with Deep Venous Thrombosis.
Authors: Kahn Susan R. , Lamping Donna L. , Ducruet Thierry , Arsenault Louise , Miron Marie Jose , Roussin Andre , Desmarais Sylvie , Joyal France , Kassis Jeannine , Solymoss Susan , Desjardins Louis
Published: October 2019

Objectives: To assess the validity and reliability of VEINES-QOL/Sym, a patient-reported questionnaire, for the evaluation of quality of life and symptoms in patients with deep venous thrombosis.

Study design: Psychometric study within the Venous Thrombosis Outcomes (VETO) Study, a Canadian multicenter prospective cohort study of long term outcomes after venous thrombosis.

Subjects: 359 English and French-speaking patients with deep vein thrombosis consecutively recruited at seven hospitals in the province of Quebec, Canada.

Outcome measures: The 26-item VEINES-QOL/Sym is a patient-reported questionnaire that we originally developed and validated for use in patients with chronic venous disorders of the leg (J Vasc Surg 2003). VEINES-QOL/Sym generates separate summary scores for symptoms and quality of life. We performed a comprehensive psychometric evaluation of the acceptability, reliability, validity and responsiveness of VEINES-QOL/Sym in the VETO study population, all of whom had objectively diagnosed symptomatic deep venous thrombosis (mean age 56 years; 50% male; 67% outpatients; 55% proximal DVT).

Results: Criterion-standard psychometric tests confirmed the acceptability (missing data, item endorsement frequencies, floor and ceiling effects), reliability (internal consistency, item-total and inter-item correlations, test-retest), validity (content, construct, convergent, discriminant, known groups) and responsiveness to clinical change of the VEINES-QOL/Sym.

Conclusions: Our results indicate that VEINES-QOL/Sym is a practical, valid and reliable measure of quality of life and symptoms in patients with deep venous thrombosis. VEINES-QOL/Sym provides a rigorous tool to allow more comprehensive evaluation of patient outcomes in clinical trials and epidemiological studies of patients with deep venous thrombosis.
First Cycle Risk of Severe and Febrile Neutropenia in Cancer Patients Receiving Systemic Chemotherapy: Results from a Prospective Nationwide Study.
Authors: Crawford Jeffrey , Wolff Debra A. , Culakova Eva , Poniewierski Marek S. , Selby Caleb , Dale David C. , Lyman Gary H. ,   for the ANC Study Group
Published: October 2019

Introduction: Myelosuppression represents the major dose-limiting toxicity of cancer chemotherapy. This prospective, nationwide study was undertaken to better define risk of neutropenia and its complications and to develop risk models for selecting patients for appropriate supportive care.

Methods: More than 3,500 patients initiating a new chemotherapy regimen have been prospectively registered at 137 randomly selected practice sites. Major disease sites include breast, lung, colorectal, ovary and lymphoma. Primary outcomes include severe neutropenia (SN; absolute neutrophil count nadir <500) and febrile neutropenia (FN) (fever/infection and nadir <1000) by cycle of treatment and overall.

Results: This analysis is based on the first 2,361 patients treated to date including 924 (39%) age ≥65. Severe neutropenia was documented in 28% and FN in 16% across all cancer categories. The proportion of patients with ANC <500 over the first four cycles of treatment include: colorectal (8%); lung (18%); ovary (23%); lymphoma (27%) and breast (37%). Likewise, the proportion of patients with FN during the first four cycles include: colorectal (7%); lung (11%); ovary (13%); lymphoma (18%) and breast (20%). As shown in the table, approximately half of the initial episodes of ANC <500 and FN over four cycles of treatment occurred in cycle 1.

Neutropenic Events By Cycle (% Patients) Primary Outcome Cycle 1 (N=2361) Cycle 2 (N=2032) Cycle 3 (N=1724) Cycle 4 (N=1246) Total (N=2361) Any SN 16 11 10 11 48 1st Cycle SN 16 6 4 2 28 Any FN 8 6 5 1 20 1st Cycle FN 8 4 3 1 16

The proportion of initial episodes of severe neutropenia that occur in cycle 1 include: colorectal (67%); lung (59%); ovary (44%); lymphoma (62%) and breast (70%). Likewise, the proportion of initial episodes of FN occurring in cycle 1 include: colorectal (53%); lung (60%); ovary (57%); lymphoma (56%) and breast (58%).

Conclusions: Rates of severe and febrile neutropenia vary with tumor type. The initial episode of severe or febrile neutropenia is most likely to occur during the first cycle of chemotherapy across all major cancer types.
Prophylactic Colony-Stimulating Factors in Pediatric Patients Receiving Myelosuppressive Chemotherapy: A Meta-Analysis of Randomized Controlled Trials.
Authors: Wittman Brenda , Horan John T. , Lyman Gary H.
Published: October 2019

Background: The colony-stimulating factors (CSFs) are widely utilized to prevent neutropenic complications in both adults and children, but randomized controlled trials in the pediatric setting have varied considerably in size, methodology and patient population. A systematic review and meta-analysis was conducted to more definitively assess the impact of prophylactic CSFs on the risk of febrile neutropenia (FN) in pediatric oncology patients. The rates of documented infection (DI) and duration of severe neutropenia (ANC <500), hospitalization and antibiotic use were also evaluated.

Methods: MEDLINE and EMBASE were searched and identified references were hand-searched through July 2004 for randomized controlled trials (RCTs) of prophylactic G-CSF or GM-CSF in pediatric oncology patients. Both parallel and crossover studies were included. Of the 625 articles identified, 16 studies were included. Ten studies evaluated children with acute leukemia or high-grade lymphoma, and 6 evaluated those with solid tumors. Study objectives, quality, and outcomes were extracted by 2 independent reviewers. Weighted summary estimates of relative risks (RR) for FN and DI were calculated by the method of Mantel-Haenszel using a fixed effects model. Mean differences in durations (days) were estimated by the method of Cohen using a random effects model due to significant heterogeneity. An economic analysis based on cost minimization was calculated from the results of this analysis and a recent cost study.

Results: Among the 12 reporting RCTs, FN occurred in 68% of 400 controls and 59% of 404 CSF patients. Estimated RR was 0.88 [.81–.97; P=.01] favoring the CSFs for leukemia and high grade lymphoma studies and 0.71 [.51–.97; P=.03] for solid tumor studies. RR estimates were 0.85 [.78–.94; P<.001) for G-CSF and 0.83 [.47–1.49; P=.54] for GM-CSF. In 9 reporting studies, DI occurred in 25% of controls and 20% of CSF patients for an estimated RR of 0.80 [.61–1.06; P=.12]. The mean decrease in duration of neutrophils <500 in 13 reporting studies was 3.5 days [2.2–4.7; P<0.0001). Mean decreases favoring CSF use were also observed for hospital stay of 1.7 days [0.9–2.5] in 13 reporting RCTs (P<.01) and antibiotic use of 2.0 days [0.4–3.6; P=.02] in 10 RCTs. Baseline estimates based on the economic model provide incremental cost savings with CSF of $3,824 per subject and a cost saving FN risk threshold of 24%.

Conclusions: This analysis reveals that prophylactic CSFs significantly decrease the incidence of FN and the durations of severe neutropenia, hospitalization, and antibiotic use in pediatric patients receiving myelosuppressive chemotherapy. Differences in rates of documented infection were not statistically signficant. The economic analysis supports guidelines for CSF prophylaxis above a risk of 20%.
Quantitative PCR Predicts Early Relapse in Patients with Chronic Lymphocytic Leukemia (CLL) Submitted to Autologous Stem Cell Transplantation. The Case for Early Clinical Intervention.
Authors: Moreno Carol , Villamor Neus , Colomer Dolors , Esteve Jordi , Bosch Francesc , Lopez-Guillermo Armando , Campo Elias , Montserrat Emili
Published: October 2019

Autologous stem cell transplants (auto-SCT) are increasingly performed in patients with CLL. Although this procedure results in a high complete response (CR) rate, most patients eventually relapse. The median time to relapse is around 5 years. Increasing levels of minimal residual disease (MRD), detected by PCR or flow cytometry (FC) are associated with clinical relapse. The early detection of patients likely to relapse shortly after SCT may be useful in the management of these patients. With this background, we analyzed the levels of MRD and its correlation with the risk of relapse and clinical outcome in 19 patients with CLL submitted to auto-SCT. MRD was assessed by FC and quantitative real time PCR of the IgH region using allele-specific oligonucleotides (ASO-PCR) in peripheral blood and/or bone marrow DNA samples obtained before SCT and at different time points thereafter. After SCT, 17 patients achieved CR and 2 partial response. A continuous pattern of relapses was observed and, after a median follow-up of 48 months (range, 11–101), 11/19 patients have progressed. The median number of CLL cells detected prior to SCT was 2.4x10−2 decreasing to 5.31x10−4 at 3–6 months after auto-SCT. No further decrease was observed beyond that point. At 3–6 months after auto-SCT, only 3/17 patients in CR had undetectable levels of disease. Patients with a MRD level >10−3 at this time point (3–6 months after transplant) had a significantly higher risk of progression than those who had less than 10−3 CLL cells. All but one patients with MRD>10−3 have relapsed (7/8) whereas only 4/9 with MRD<10−3 did so. As shown in the figure, median time to progression was significantly shorter in those patients with a higher MRD level (16 vs. 55 months; p=0.003)

Figure Figure

In conclusion, quantification of MRD within the first 6 months after auto-SCT allows the identification of CLL patients with a high risk of early clinical relapse. These data provide background to investigate whether early treatment, before clinically overt relapse occurs, might be useful in patients with high risk of relapse after SCT.
Acquirement of Rituximab Resistance in Lymphoma Cell Lines Is Associated with Structural Changes in the Internal Domain of CD20 Regulated at the Post-Transcriptional Level.
Authors: Czuczman Myron S. , Olejniczak Scott , Gowda Aruna C. , Starostik Petr , Hernandez-Ilizaliturri Francisco J.
Published: October 2019

The acquirement of resistance to rituximab has been observed in lymphoma patients. To further define the molecular basis for rituximab-resistance we have developed various rituximab-resistant cell lines (RRCL) and studied changes in CD20 structure at the protein and gene level, membrane reorganization, and signaling events following rituximab exposure. RRCL were generated by chronic exposure of Raji cells to escalating doses of rituximab alone (2R) or concurrently with human complement (4RH). Functional assays were performed to demonstrate decrease in rituximab-associated CMC and ADCC. Changes in the structure of CD20 were determined by Western blotting using various antibodies recognizing epitopes located in the internal (GST77 and 1439) and external domain (B1) of CD20. Sequencing of the CD20 gene from Raji parental and RRCL was performed to determine differences. Following rituximab treatment, lipid raft domains were extracted from Raji and RRCL to determine changes in polarization. Redistribution of CD20 antigen were evaluated by Western blotting. No significant changes were observed in the expression of the external domain of CD20 antigen between rituximab-sensitive and RRCL as demonstrated by flow cytometric analysis and Western blotting. However, significant changes in the internal domain of CD20 were observed in RRCL. Specifically, changes in the N-terminal, and to a lesser degree the C-terminal region of the internal domain of CD20 were observed in RRCL. The CD20 gene sequence was found to be identical between Raji cells and RRCL, suggesting a post-transcriptional regulatory mechanism is responsible for the changes in the structure of CD20. Redistribution of CD20 into the lipid raft domains was more evident in Raji parental cells when compared to RRCL, and a decrease in p38 activation was observed following exposure of RRCL to rituximab. In conclusion, our data strongly suggests that the acquirement of rituximab-resistance is associated with significant changes in the structure of the internal domain of the CD20 antigen. The abnormal production of truncated forms of the CD20 antigen, while not affecting external rituximab binding resulted in changes in the redistribution of CD20 antigen into lipid raft domains and a decrease in signaling events. Post-transcriptional events leading to the acquirement of truncated forms of CD20 may impair signaling activation, thus contributing to rituximab-resistance seen in RRCL.