bluuebunny/crossref_metadata_2025 huggingface dataset:
The Promising Strategy of Mycophenolate Mofetil Dosing to Prevent Moderate- to Severe-Acute Graft-Versus-Host Disease.
Authors: Nishikawa Shinichiro , Okamura Atsuo , Yamamori Motohiro , Katayama Yoshio , Shimoyama Manabu , Matsui Toshimitsu
Published: April 2020

To prevent acute graft-versus-host disease (GVHD) and graft failure, mycophenolate mofetil (MMF) has been employed as a substitute of methotrexate in allogeneic hematopoietic stem cell transplantation (all-SCT). However, the dosing strategy of MMF after all-SCT remains to be established.

In this study, we investigated the optimal MMF dosing until day 30 based on pharmacokinetic studies in 28 Japanese allo-SCT patients. For better clinical outcomes of MMF, higher mycophenolic acid (MPA) plasma levels are proposed to be desirable. Therefore, the first 11 patients (group A) received MMF orally every 12 hours at an escalated dose from 15 mg/kg to 25 mg/kg (maximum total daily dose 3000 mg), according to real-time pharmacokinetic monitoring of the total MPA area under the curve (AUC). However, the dose escalation in each individual did not always increase the AUC. Then, MMF was given orally at a fixed dose of 1000 mg every 8 hours in the subsequent 17 patients (group B). The pharmacokinetic data revealed that the increase of dosing frequency could statistically keep higher MPA plasma levels, as reflected in concentration at steady state (Css: group A vs. group B; 1.12 vs. 2.18 μg/ml on day 16, P = 0.003) or trough value (Ctrough: 0.18 vs. 0.56 μg/ml on day 16, P = 0.019). These results indicate that MMF administration of every 8 hours would be better than that of every 12 hours even in the same total daily dose until day 30.

We further assessed the safety and efficacy of extended MMF administration beyond day 30 retrospectively. Twenty-five patients ceased MMF at day 30, whereas 16 patients were subjected to extended regimen depending on the individual risk factors for GVHD (median dosing period 64.5 days, 50–94 days). No severe adverse events were observed in both groups. While the cumulative incidence (CI) of more than grade I acute GVHD at day 100 was comparable between the two groups (72.1 % vs. 62.5 %, P = 0.63, Figure 1A), the CI of grade II to IV acute GVHD was lesser in the latter group (42.3 % vs. 12.5 %, P = 0.045, Figure 1B).

These results suggest that MMF should be administered every 8 hours until day 30 after allo-SCT, followed by extending MMF administration at least until day 60 as a preemptive therapy for moderate- to severe-acute GVHD. This strategy might be very useful for double UCB transplantation under non-myeloablative conditioning especially, because the transplantation is reported to develop severe acute GVHD frequently.

Figure Figure
Re-Evaluation of Chronic Graft-Versus-Host Disease Using National Institute of Health Consensus Criteria.
Authors: Kim Dae-Young , Lee Je-Hwan , Lee Jung-Hee , Kim Se Hyung , Lim Sung-nam , Lee Young-Shin , Kang Young-Ah , Kang Sol-Ip , Seol Miee , Ryu Seong-Gil , Lee Kyoo-Hyung
Published: April 2020

Background: The National Institutes of Health (NIH) proposed new consensus criteria for the diagnosis and scoring of chronic graft-versus-host disease (cGVHD). Using the new system, we re-evaluated the patients with cGVHD that was diagnosed by classic criteria.

Methods: Of 618 patients who underwent allogeneic hematopoietic cell transplantation (HCT) from December 1994 to April 2008 at the Asan Medical Center, Seoul, Korea, we retrieved 236 patients who had cGVHD by classic criteria from the AMC BMT Registry. Among 236 patients, 20 patients with liver-only involvement could not be diagnosed as cGVHD by the NIH criteria, thus we reclassified and graded 216 patients according to the NIH criteria. We also evaluated the ability of the NIH criteria to stratify and predict the risk of cGVHD patients, as assessed by GVHD-specific survival (GSS).

Results: Twenty patients (9.3%) were reclassified as acute GVHD by NIH criteria (‘classic acute’ in 7 and ‘persistent, recurrent, or late-onset acute’ in 13) and 196 patients (90.7%) remained as chronic GVHD (‘classic chronic’ [Cl-Ch] in 170 and ‘overlap syndrome’ [Ov-Sy] in 26). Median age of 196 patients with cGVHD by NIH criteria, 119 males and 77 females, was 35.5 years (range, 15 to 57). Acute GVHD preceded cGVHD in 70 patients (35.7%). The probability of GSS at 5 years was 86.2% with 22 cGVHD-related deaths. The GSS was significantly different between two subtypes of cGVHD by NIH criteria: 88.6% for Cl-Ch vs 70.2% for Ov-Sy (p=0.002). NIH global scoring system stratified risk of cGVHD patients better than stage by classic criteria at both onset and peak of cGVHD (Table 1). We evaluated 12 variables at onset of cGVHD to determine their prognostic significance for GSS. Multivariate analysis demonstrated that NIH global score at onset (mild vs moderate, HR 6.1, p=0.027; mild vs. severe, HR 7.0, p=0.015), preceding aGVHD (no vs. yes, HR 6.2, p=0.001), and number of HLA ABDR mismatch (0 vs. 1, HR 2.0, p=0.555; 0 vs 2, HR 200.4, p=0.009) were independent predictors for GSS.

Conclusion: Our results indicate that a new NIH system can provide a proper risk-stratification of patients with cGVHD and global scoring system at onset of cGVHD can predict the prognosis of patients.

Table 1. GVHD-specific survival according to NIH global scoring system or stage by classic criteria

Onset of cGVHD Peak of cGVHD Pt. No. GSS (5-y) P-value Pt. No. GSS (5-y) P-value NIH criteria mild 70 95.2% 0.022 36 100% 0.004 moderate 64 82.8% 42 92.2% severe 62 79.7% 118 79.6 Classic criteria limited 86 90.1% 0.305 45 97.8% 0.039 extensive 110 83.7% 151 83.0%
Silencing of Mir-22 Is Linked to H3K27 Trimethylation and Independent of DNA Methylation in Pre-B Acute Lymphoblastic Leukemia Cells.
Authors: Li Xiaoqing , Liu Jun , Zhou Rui , He Yanli , Huang Shi , Huang Shiang , Chen Xian-Ming
Published: April 2020

The regulation of human microRNA (miRNA) expression is still poorly understood and aberrant epigenetic regulation has recently been implicated in the down-regulation of tumor suppressor miRNAs. In this study, we investigated whether histone modifications would contribute to the dysregulation of miRNAs in lymphoblastic leukemia cells. Using a precursor B-cell acute lymphoblastic leukemia cell line, NALM-6 cells, we demonstrated by miRNA microarray analysis that a specific histone deacetylases inhibitor, trichostatin A (TSA), induced a differential alteration in cellular miRNA expression. A total of 10 miRNAs were down-regulated and 31 up-regulated significantly following TSA treatment. Among TSA-up-regulated miRNAs, miR-22 is an extronic miRNA and resides in the second exon of the non-coding transcript MGC14376. Up-regulation of both miR-22 and MGC14376 was found in NALM-6 cells treated with TSA but not 5-AZA-2’-deoxycytidine, a DNA demethylating agent. Luciferase reporter analysis identified three regions in the promoter of miR-22 and MGC14376 that differentially regulated its transcriptional activation. Although there is a CpG island within the promoter of miR-22 and MGC14376, no obvious methylation was detected at this region in NALM-6 cells. Conversely, H3K27 trimethylation (H3K27triM)-associated histone modification was identified in the first intron of MGC14376 gene and was involved in TSA-induced miR-22 expression. Thus, miR-22 silencing in NALM-6 cells involves H3K27triM-associated histone modification but is independent of DNA methylation, suggesting that methylation-independent H3K27triM histone modification may be an important mechanism for miRNA dysregulation in cancer cells.
Acute Myeloid Leukemia Is Characterized by Wnt Pathway Inhibitor Promoter Methylation.
Authors: Griffiths Elizabeth A. , Hooker Craig M. , McDevitt Michael A. , Karp Judith E. , Herman James G. , Carraway Hetty E.
Published: April 2020

The Wnt pathway contributes to a stem-cell like phenotype in a variety of cancer subtypes. Nuclear localization of non-phosphorylated, active β-catenin is a surrogate marker for Wnt pathway activation and has been associated with adverse outcome in patients with acute myeloid leukemia (AML). Wnt pathway inhibitors including APC, DKK1, DKK3, LKB1/STK11, RASSF1A, RUNX3, SFRP1, SFRP2, SFRP4, SFRP5, SOX17, and WIF1 contain extensive promoter region CpG islands. Wnt pathway inhibitors are silenced by promoter methylation in many malignancies including lung cancer, colon cancer and acute lymphoid leukemias. To determine if methylation of these Wnt pathway inhibitors is present in AML, we evaluated leukemia cell lines (i.e., K562, HNT34, KG1, KG1A, U937, and HL60) for evidence of promoter methylation. Additionally, 188 AML patient (median age 61 years) samples from the Johns Hopkins Hospital leukemia tumor bank were assessed for the presence of Wnt pathway inhibitor methylation. All samples were bisulfite treated and evaluated for promoter methylation using methylation specific PCR. Diagnostic samples from a subgroup of patients with normal cytogenetics (n=73) who received high dose induction therapy were evaluated for potential associations between methylation of individual Wnt pathway inhibitor genes or total number of methylated genes and event free or overall survival.

RESULTS: Extensive promoter methylation of the Wnt pathway inhibitor genes was observed in leukemia cell lines. Of the primary leukemia samples, 85% had at least one methylated gene. Promoter methylation of Wnt inhibitors was common in these samples with the following frequencies: DKK1 (16%;30/188), DKK3 (8%;15/188), RUNX3 (27%;50/188), SFRP1 (34%;63/188), SFRP2 (66%;124/188), SFRP4 (9%;16/188), SFRP5 (54%;102/188), SOX17 (29%;54/188), and WIF1 (32%;61/188). This is among the first comprehensive evaluations of Wnt pathway inhibitor methylation in primary samples from AML patients. The frequency of methylation seen here is comparable to that observed in established tumor suppressor genes in patients with AML, including p15INK4B, SOCS1 and CDH1. In marked contrast with epithelial tumors, methylation of APC (2%;2/108) and RASSF1A (0%;0/188) was rare. LKB1/STK11 methylation was also uncommon (2%;2/108). Previous reports have associated Wnt pathway activation with poor prognosis. In our treated patients with normal cytogenetics, no correlation was observed between methylation of Wnt pathway inhibitors and event free or overall survival. In conclusion, in patients with AML (a) there is a high frequency of Wnt pathway inhibitor methylation; (b) Wnt pathway inhibitor methylation is distinct from that observed in epithelial malignancies; and (c) methylation of Wnt pathway genes does not correlate with adverse clinical outcome.
Pathogenesis of Acute Myeloid Leukemia: In Vitro Studies Using Primary Human Cells.
Authors: Abdul-Nabi Anmaar , Yassin Enas R , Takeda Akiko , Yaseen Nabeel R.
Published: April 2020

Many cases of acute myeloid leukemia (AML) are associated with non-random chromosomal rearrangements and most of these result in fusions involving retinoic acid receptor α (RARα), CBF transcription factors, MLL, and nucleoporins. Here, we report the effects of key members of these four major groups of AML-associated chimeric fusion proteins on differentiation, proliferation, self-renewal and gene expression in primary human CD34+ hematopoietic cells. We expressed the PML-RARα, AML1-ETO, MLL-AF9 and NUP98-HOXA9 fusion genes in human peripheral blood CD34+ cells by retroviral transduction and compared them to cells transduced with empty vector. By colony forming assays, morphological examination and flow cytometric immunophenotyping we found that PML-RARα causes some degree of myeloid differentiation block. AML1-ETO had no obvious effect on differentiation, while MLL-AF9 and NUP98-HOXA9 caused a block in both myeloid and erythroid differentiation. All fusion oncoproteins, except PML-RARα, induced dramatic long-term proliferation in liquid culture and a marked increase in the numbers of primitive long-term culture-initiating cells (LTC-ICs). In order to understand the molecular basis of these effects, gene expression profiling was performed for each fusion gene by oligonucleotide microarray analysis at 3 different time points (6 h, 3 days, and 8 days post transduction). At the 6 h time point both AML1-ETO and PML-RARα caused extensive changes in gene expression with a predominance of downregulated genes. The number of genes dysregulated by AML1-ETO and PML-RARα decreased dramatically at the 3-day time point. These data are consistent with the fact that these two oncoproteins are DNA-binding transcription factors that are known to repress transcription. In contrast, MLL-AF9 and NUP98-HOXA9 altered the expression of fewer genes at the 6 h time point with a preponderance of upregulated genes; at the 3 day time point, the number of genes dysregulated by NUP98-HOXA9 and MLL-AF9 increased. These findings suggest that NUP98-HOXA9 and MLL-AF9 may have delayed effects that are not due to direct transcriptional regulation. Homeobox transcription factors were upregulated by both MLL-AF9 and NUP98-HOXA9, but not by either AML1-ETO or PML-RARα. The results of the biological assays and gene profiling show marked similarities between NUP98-HOXA9 and MLL-AF9, and suggest that they transform cells by similar pathways that are different from those used by AML1-ETO and PML-RARα.
NF-κB Is Active in Mcl-1 Promoter Regulation in Human CLL.
Authors: Hertlein Erin K , West Derek A. , Craig Ruth W , Lucas David M , Byrd John C.
Published: April 2020

The NF-κB family of transcription factors is linked to neoplasia due to its role in increasing cell proliferation as well as inhibiting apoptosis. NF-κB has been reported to be constitutively active in chronic lymphocytic leukemia (CLL), but the mechanism underlying this this activation is not fully understood and the critical target genes involved have not been identified. To further define the mechanism(s) by which NF-κB promotes survival in CLL, we investigated the role of this transcription factor in the regulation of the myeloid cell leukemia 1 (Mcl-1) gene. The promoter region of Mcl-1 contains a putative NF-κB binding site, and increased expression of the Mcl-1 protein has been related to rapid disease progression and resistance to apoptosis in CLL. However, a direct link between NF-κB and Mcl-1 transcriptional regulation has not yet been established in B-cells or human CLL. We demonstrate here that NF-κB binding is a critical event in the transcriptional regulation of Mcl-1, as deletion of the NF-κB binding site in the promoter results in decreased activity of an Mcl-1-luciferase reporter construct. In addition, pharmacological inhibition of NF-κB reduced p65 nuclear localization and binding of NF-κB to the Mcl-1 promoter. Interestingly, when CLL patient samples were exposed to an IKK inhibitor, Bay-11, the extent of Mcl-1 inhibition varied in patients. We therefore examined whether the effect on Mcl-1 correlated with in vitro cell survival, a result that might be expected given that NF-κB expression has been previously reported to correlate with CLL cell survival. We found that CLL patient cells more sensitive to Bay-11-induced apoptosis also showed a larger decrease in Mcl-1 mRNA, suggesting that Mcl-1 message level may be a useful diagnostic to predict patients that will respond to NF-κB targeted therapy. Furthermore, different inducers of the NF-κB signaling pathway (CD40L, immune stimulatory CpG-ODN and TNF-α), promote differential effects on Mcl-1 regulation in CLL patient cells. Although all three treatments increase NF-κB nuclear localization and DNA binding, Mcl-1 RNA and protein increased with CpG and CD40L treatment, but not TNFα. This result provides evidence of stimulus-specific regulation of Mcl-1 by NF-κB. Based on these observations, we hypothesize that there is a direct link between NF-κB DNA binding and transcriptional control of Mcl-1, and that this gene could serve as a pharmacodynamic endpoint to monitor the efficacy of NF-κB inhibitors in CLL cells. Additionally, a direct link of NF-κB activity to Mcl-1 expression and cell survival provide further justification for targeting this transcription factor for treatment in CLL.
Impact of Rituximab on Peripheral Blood Stem Cell Mobilization and Collection Following ACVBP Regimen in Poor Risk Patients with Diffuse Large B-Cell Lymphoma (DLBCL): Results from a Large Cohort of Patients from Two Prospective GELA Trials.
Authors: Lefrere Francois , bastit-Barrau Dominique , Mathieu Suzanne , Bohbot Alain , Bourrin Philippe , Salles Gilles Andre , Tilly Herve , Mounier Nicolas , Fitoussi Olivier , Haioun Corinne
Published: April 2020

<>The ACVBP regimen is commonly used in young poor-risk patients with DLBCL candidates to first line consolidative high-dose therapy followed by autologous stem cells transplantation in GELA trials. The combination with the monoclonal anti-CD20 antibody rituximab (R-ACVBP) is now routinely used, as induction treatment and to mobilize peripheral blood stem cell (PBSC). The aim of the present study was to assess the impact of rituximab on PBSC mobilization and collection in patients with newly diagnosed DLBCL receiving ACVBP chemotherapy. We reviewed the data from two prospective controlled trials. The first, conducted between 1999 and 2003, involved patients presenting with 2 or 3 adverse prognostic factors on the basis of the age-adjusted IPI (aa-IPI), treated by ACVBP (LNH 98B-3) (ASCO2007:8018). In the second trial (LNH 03-3B), conducted between 2004 and 2007, similar patients received the same initial inductive chemotherapy combined to rituximab (375mg/m2 at D1). 137 and 91 patients in the ACVBP and the R-ACVBP groups are here analyzed, respectively. Clinical and biological characteristics at diagnosis of the two groups of patients were similar (aa-IPI 2 and aa-IPI 3: 75% and 25%, respectively). The conditions for G-CSF administration and stem cell collections were identical. PBSC mobilizations were performed following the third or fourth cycle of (R)-ACVBP. The median delay between day 1 of chemotherapy and the first hemapheresis was identical for both groups. First hemapheresis was performed with a median peripheral white blood cell concentration of 16.2 x 109/l and 16.6 x 109/l for ACVBP and R-ACVBP groups, respectively. The median peak number of peripheral blood CD34+ cells observed the same day of first hemapheresis in ACVBP and R-ACVBP group was 69 x 106/l and 63 x 106/l, respectively (p = 0.5). The median number of CD34+ cells collected were 7.1 x 106 and 6.0 x 106 CD34 cells/kg for ACVBP and R-ACVBP groups (p = 0.12) while the median number of hemapheresis to target a minimal number of 3 x 106 CD34 cells/kg was identical, of one, in both groups. Failure of stem cell collection , defined as less than 3 x 106 CD34 cells/kg harvested, was observed in 8% and 4% of the patients who received ACVBP or R-ACVBP , respectively (p = 0.13). We conclude that rituximab combined to ACVBP regimen does not impair PBSC mobilization and collection.
The Antioxidant Inflammation Modulator, CDDO-Me Promotes Myelopoiesis in Mice.
Authors: Welniak Lisbeth A. , Harouna Salif , Prosser David , Ames Erik , Meyer Colin , Murphy William J.
Published: April 2020

CDDO-me (methyl-2-cyano-3,12-dioxooleana-1,9-diene-28-oate)is a novel antioxidant inflammation modulator that has been shown to suppress tumor-induced inflammatory processes through suppression of NF-kB and STAT3 and induce apoptosis and differentiation of human leukemic and solid tumor cells.. It has also been shown to be more potent than the parent compound, CDDO against leukemia cells. We have previously shown that administration of CDDO, when given peri-transplant, can inhibit the development of acute murine GVHD and induces cell death in proliferating but not resting lymphocytes in a mixed lymphocyte culture. We therefore next sought to determine the effect of CDDO-me administration on normal hematopoiesis in naive and myelosuppressed mice. Administration of CDDO-me (120 ug/dose BID for 7 days) to C56BL/6 mice did not significantly affect the number of T and B cells in the spleen. Unexpectedly, CDDO-me administration resulted in a significant expansion of myeloid cells in the bone marrow and spleen, predominately CD11b+Ly6CintGR1hi neutrophils. To determine if CDDO-me acted primarily on terminal stages of differentiation or at early phases on development in hematopoiesis, we performed clonogeneic assays for myeloid (CFU-GM), erythroid (BFU-E) and primitive, high proliferative potential (CFU-HPP) precursors and compared treatment with CDDO-me with rhG-CSF administration, a cytokine known to promote expansion and mobilization of hematopoietic precursors from the bone marrow. Administration of CDDO-me resulted in significant expansion of myeloid and erythroid progenitors in the spleen and blood although not to the same extent as that observed in mice receiving 2.5 ug/dose BID rhG-CSF. Unlike the mobilization seen with rhG-CSF where hematopoietic precursors were decreased in the bone marrow, CDDO-me significantly increased the number of myeloid, erythroid and CFU-HPPs in the marrow. Administration of CDDO-me prior to myelosuppression with sublethal total body irradiation also resulted in significant increase of spleen cellularity after 8 days recovery due to significant expansion of myeloid cells as well as increases in B cells. However, CDDO-me did not significantly impact hematopoietic recovery compared to vehicle control treated animals when CDDO-me was given to mice following sublethal total body irradiation. These results suggest that CDDO-me can expand early hematopoietic precursors in mice, resulting in radioprotection. Stimulation of myelopoiesis in vivo by CDDO-me can be accounted for, in part, by a significant increase in circulating G-CSF levels in treated animals. However, the retention of hematopoietic precursors in the bone marrow of mice that received CDDO-me but not rhG-CSF suggest additional mechanisms are involved. These results demonstrate that CDDO-me significantly affects normal hematopoietic cells including precursor populations in vivo and can promote granulopoiesis predominately in non-irradiated mice.
UTY-Coded Minor Histocompatibility Antigens as Targets for a Graft- Versus-Host Reaction against Hematopoietic Cells in the Dog.
Authors: Bund Dagmar , Buhmann Raymund , Zorn Julia , Schmetzer Helga , Kolb Hans- Jochem
Published: April 2020

Female donors produce a strong graft-versus-leukemia (GvL) effect in male patients. Presumably Y-encoded minor histocompatibility antigens (mHA) are recognized by the alloimmune effector lymphocytes. UTY (ubiquitously transcribed tetratricopeptide repeat, Y-linked) is a potential candidate gene for mHA in dogs and men. In the canine model we could show that the minor histocompatibility antigen UTY might be a potential target to further improve the adoptive immunotherapeutic approach. Five HLA-A2 restricted peptides were used for pulsing canine dendritic cells and stimulating canine T cells. T-cells were separated from the blood of female donors using the MACS technique and effector T-cells were generated by incubation of 2x10*6 CD3+ T-cells/ml with autologous mature, peptide-pulsed dendritic cells (DCs) in X-Vivo 15 medium (+ 160 U/ml human IL-2 and 5 ng/ml human IL-7; ratio T:DCs=10:1). T cells were restimulated weekly with peptide-pulsed DCs. After 3 or 4 weeks in culture T-cells were examined for the CD8/CD4 ratio, cytotoxicity assays were assessed after 3–4 re-stimulations in [51Cr]-release assays. The number of peptide-specific T cells was assessed in IFN-g-ELISPOT-assays. UTY-mRNA expression was determined by RT-PCR in several female and male cell types (BM, PBMCs, monocytes, DCs, B-cells). We were able to expand T-cells from 6 female dogs using peptide-pulsed autologous DCs; 3 out of 5 UTY-derived peptides were able to stimulate a specific autologous T-cell response. The number of CD8+ T-cells could be expanded during four weeks of in vitro culture in 3 dogs. In IFN-g-ELISPOT-assays we could demonstrate that the expanded T-cells secrete IFN-g upon recognition of DCs from DLA-identical male littermates. In addition, these T-cells specifically lysed DLA-identical male BM (25-100%), but not autologous or DLA-identical female BM (0–15%) in [51Cr]-release assays (E:T=80:1). Cell types other than BM and DCs (DLA-identical male and female monocytes, PBMCs, B-cells) were not lysed (0–18%). In summary, we could demonstrate that UTY is a naturally processed and presented antigen in the dog. Our data show that HLA-A2 restricted UTY-peptides immunize specific CTLs from female dogs that efficiently recognize DLA-identical male bone marrow and DCs. These findings suggest that UTY may be employed for studies of immunotherapy in the canine model.
GITR Ligand on Leukemic Myeloid Dendritic Cells Suppresses Induction of Leukemia-Associated Antigen-Specific CTLs from Naïve CD8+ T Cells.
Authors: Kondo Yukio , Katagiri Takamasa , Ohata Kinya , Nakao Shinji
Published: April 2020

Glucocorticoid-induced TNFR-related protein (GITR), a member of the TNF receptor superfamily, is expressed at low levels on resting T cells and is up-regulated following activation. Triggering of GITR with its ligand (GITRL) has been described to abrogate the function of CD4+CD25+ regulatory T cells and stimulate effector T cells in mice, but little is known about roles of GITR-GITRL interaction in humans. Recent evidence suggests that the interaction between GITR on NK cell and GITRL, which is constitutively expressed on tumor cells, negatively regulates NK cell-mediated anti-tumor activity in cancer patients. Leukemic myeloid dendritic cells (mDCs) can induce cyclin-dependent kinase (CDK2)- specific cytotoxic T lymphocytes (CTLs) from naïve T cells and CDK2-specific CTLs are detectable in MRD+ patients with leukemia after allo-SCT (Blood. 110 (11): a3228. 2007). The GITR-GITRL interaction may affect this process and a modification of the interaction may improve the efficiency of inducing CDK2-specific CTLs. Therefore, the expression of GITRL on leukemic cells and leukemic mDCs was examined to determine whether an engagement of GITR controls the priming of leukemia-associated-antigen (LAA)-specific CD8+ T cells. When cryopreserved BMMCs obtained at diagnosis from 5 patients with AML were assessed using flow cytometry, GITRL expression was detectable on leukemic cells in 3 patients. Leukemic mDCs were enriched from PBMCs of HLA-A24+ patients with anti-CD1c mAb-conjugated magnetic beads and were assessed for their ability to stimulate HLA-A24+ naïve CD8+ T cells to acquire cytotoxicity specific to CDK2-peptides (CDK2 158–166, CDK2 178–186). A three days culture of immature leukemic mDCs in the presence of TNFα up-regulated the expression of GITRL along with CD83 and CD40 expression. Naïve CD8+ T cells isolated from healthy individuals and cord blood were cultured with the GITRL-expressing leukemic mDCs in the presence or absence of anti- GITR monoclonal antibodies (mAbs) for 14 days and stained for CDK2 158–166/A24, CDK2 178–186/A24 pentamers. Blocking of GITR with the mAbs augmented induction of CDK2 158–166- and CDK2 178–186- specific CD8+ T cells from 0.37% to 1.17% and from 0.45% to 1.64%, respectively (Fig). Anti-GITR mAbs did not enhance induction of CDK2-specific T cells by peptide-pulsed monocyte-derived DCs which do not express GITRL. These data suggest that the expression of GITRL on circulating leukemic mDCs may suppress induction of CTLs specific to LAAs and induce cancer immunoediting in patients with leukemia. Administration of anti-GITR mAb after allo-SCT may enhance graft versus leukemia effect by CDK2-specific CTLs without vaccination of CDK2-peptides.

Fig Blocking of GITR on T cells augments induction of CDK2-specific CTLs Fig. Blocking of GITR on T cells augments induction of CDK2-specific CTLs
Indirect Comparison of the Efficacy of Melphalan-Prednisone-Bortezomib Relative to Melphalan-Prednisone-Thalidomide and Melphalan-Prednisone for the First Line Treatment of Multiple Myeloma
Authors: Yeh Yating , Chambers James , Gaugris Sabine , Jansen Jeroen
Published: April 2020

Melphalan-prednisone (MP) combination has been considered a standard of care for front line treatment of multiple myeloma in patients non eligible for transplant. Melphalan-prednisone-bortezomib (MPV) combination has been approved in the United States in patients non eligible for high-dose chemotherapy (HD-C) and has recently received a positive opinion from the CHMP in Europe. Melphalan-prednisone-thalidomide (MPT) was approved in Europe in patients >65 or not eligible for HD-C. There is no head-to-head trial directly comparing MPV to MPT. The objective of the current study was to compare the efficacy of MPV to MP and MPT as first line treatment of multiple myeloma in patients non eligible for transplant. Six randomized placebo controlled trials investigating the efficacy of MPT (5) and MPV (1) relative to MP were identified with a systematic literature review. The endpoints of interest were overall survival (OS), progression free survival (PFS) and overall and complete response. Relative efficacy estimates of MPT versus MP as obtained from the MPT-MP trials were combined with meta-analysis techniques and simultaneously indirectly compared with the relative efficacy of MPV versus MP from the MPV-MP trial (VISTA). This adjusted indirect comparison was performed with Bayesian fixed and random effects models. As compared to frequentist approach, Bayesian meta-analysis offers a more informative summary of the likely value of efficacy after observing the data and allows for direct probabilistic inferences. Of the three interventions compared, there was an 81% probability that MPV was the most efficacious intervention in terms of overall response and a >99% probability in terms of complete response. With MPV a patient was two times more likely to show a complete response than with MPT (Relative Risk=2.15; 95%Credible Interval (CrI): 0.99–4.45). Both MPV and MPT showed greater OS than MP (HR=0.61; 95%CrI: 0.42–0.88 and HR=0.61; 95%CrI: 0.47–0.78 respectively); the indirect comparison showed similar efficacy in terms of OS between MPV and MPT (MPV vs MPT: Hazard Ratio=1.00; 95%CrI 0.64–1.57). Both MPV and MPT also displayed greater PFS than MP (MPV versus MP: HR=0.61; 95%CrI 0.49–0.76 and MPT versus MP HR=0.51; 95%CrI 0.41–0.63 respectively) and showed similar efficacy (MPV vs MPT: HR=1.19; 95%CrI: 0.87–1.63). In this study, both MPV and MPT are more efficacious than MP in terms of response, PFS and OS. MPV is expected to result in a greater complete and overall response than MPT. No difference in OS or PFS was displayed. Further analyses will need to be undertaken once evidence base data is more mature.
Quality of Life Scores Pre and Post Autologous Stem Cell Transplant: A Comparison of Patients Greater Than 60-Years-Old Vs Younger Than 60-Years-Old
Authors: Dabney Jane , Rybicki Lisa , McLellan Linda , Foster Larry , Visnosky Michele , Curtis Julie , Bolwell Brian
Published: April 2020

Prospective quality of life instruments were administered to 304 patients with non-Hodgkin’s lymphoma (NHL), Hodgkin disease (HD), and Multiple Myeloma (MM) undergoing autologous stem cell transplantation (ASCT) from June 2003 through December 2007. The two groups were fairly balanced with respect to baseline characteristics with the younger group having slightly more HD and the older group having slightly more NHL. Of the 304 total patients, 65% had NHL, 21% MM, and 14% HD; median age was 53; 60% were male; 25% received prior radiation therapy; preparative regimens were busulfan based or melphalan; all patients received peripheral stem cells alone, median inpatient length of stay was 21 days. The Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) and the Profile of Mood State (POMS) were administered to patients prior to their priming for stem cell collection and again 40–44 days (Day 42) post ASCT. The FACT-BMT measures five components: physical well being (PWB), social well being (SWB), emotional well being (EWB), functional well being (FWB), and additional concerns (AC). The POMS measures depression, vigor, anger, tension, confusion, and fatigue. In looking at the FACT-BMT the 60 + group reported better emotional well-being at baseline than the <60 group (p= 0.008) and on day 42 post transplant the 60+ group reported significantly better EWB from the under 60 group (p<0.001). Both groups report a decrease in FWB from baseline to day 42 (p=0.022) but the 60+ group reports higher FWB than the other group. Results of the POMS indicates that the <60 group reported higher depression and anger at baseline than did the 60+ group (p<0.001). The <60 group also reported greater tension (p=0.002) at baseline. Confusion was rated slightly higher at baseline also by the <60 group (p=0.020). There were no significant differences between the two groups in these areas at day 42. In conclusion, one can speculate that the 60+ group is more likely to have experienced family or personal crisis prior to their diagnosis and may have more established coping mechanisms on which to rely. Additionally, those in the <60 group may tend to have more family and employment considerations that may distract them from being able to focus on their treatment and preparation for transplant. It is possible that the <60 group would benefit from earlier involvement from the social worker to assess their needs and more specialized family oriented education and/or materials.
Granulocyte Colony-Stimulating Factor (G-CSF) Stimulates Sympathetic Nervous System Activity in the Bone Marrow
Authors: Battista Michela , Mendez-Ferrer Simon , Frenette Paul S.
Published: April 2020

Granulocyte colony-stimulating factor (G-CSF) is the agent most commonly used clinically to elicit hematopoietic stem and progenitor cells (HSPCs) mobilization from the bone marrow (BM) to the peripheral blood. Our previous studies indicate the sympathetic nervous system (SNS) controls G-CSF-induced HSPCs egress from the BM and that G-CSF decreases catecholamine levels in bone/BM tissues (Cell2006; 124:407). However the molecular basis for this depletion is unknown. Here we show that G-CSF stimulates selectively the activity of the SNS in bone/BM microenvironment in mice. Norepinephrine (NE), a catecholamine and the principal neurotransmitter of the SNS, is synthesized by the rate-limiting enzyme tyrosine hydroxylase (TH). Since TH is a catecholaminergic marker which reflects the extent of sympathetic innervation, we evaluated its expression in bone/BM tissues after G-CSF treatment. TH immunofluorescence staining analysis in longitudinal frozen sections of femurs exhibited a strong punctuate staining of the catecholamine-producing enzyme that was increased 10-fold in G-CSF-treated mice compared to control animals. This result was also confirmed by Western blot analysis of equal amount of protein obtained from BM lysates of G-CSF-treated and control animals. Because NE release and removal from the extracellular space determine tissue NE turnover and regulate the sympathetic activation, we evaluated NE release and uptake in sympathetic cervical ganglia (SCG) in organ cultures treated with G-CSF. Although the release of [3H]NE from cultured ganglia was not affected by G-CSF, we found a significant reduction in the reuptake rate. NE clearance from the synaptic cleft is mediated by the Na+-dependent NE transporter (NET). NET is selectively expressed and localized in lipid rafts of the plasma membrane of the noradrenergic nerve terminals and it is internalized as a result of its down-regulation. We thus isolated lipid rafts from BM homogenized in lysis buffer containing 1% Triton X-100 and subjected to sucrose gradient centrifugation. We found that G-CSF induced the redistribution of NET from the plasma membrane as revealed by a strong reduction of NET levels in BM lipid raft-enriched gradient fractions and by an increase of NET levels in BM non-raft fractions following G-CSF treatment. Moreover Q-PCR analysis of catecholamine-inactivating enzymes revealed reduced mRNA levels of the monoamine oxidase (MAO) and the catechol-O-methyltransferase (COMT) enzymes derived from BM of mice injected with G-CSF compared to untreated animals. Altogether, these results demonstrate that G-CSF treatment affects NE turnover impairing the removal of NE from the synaptic cleft and therefore leading to an increase of the concentration of the extracellular NE. A likely consequence is a prolongation of the postsynaptic action of NE on its receptors and ultimately an augmented sympathetic activity in the bone marrow microenvironment.
Functional analysis of the Human Pbx Interacting Protein (HPIP) in Human Hematopoiesis
Authors: Kaur Pawandeep , Stadler Christiane , Ahmed Farid , Cusan Monica , Humphries R. Keith , Hiddemann Wolfgang , Buske Christian , Feuring-Buske Michaela
Published: April 2020

The recognition of novel proteins that regulate human hematopoietic stem cell and early progenitor cell fate is a prime objective in experimental and clinical hematology. Human hematopoietic PBX interacting protein (HPIP), with no significant homology to known proteins, is a 731 amino acid protein, discovered as a novel interacting partner of the PBX homeodomain protein. HPIP has been implicated as a nuclear-cytoplasmic shuttle molecule and shown to have the capacity to bind to the cytoskeleton. It also inhibits the ability of PBX-HOX heterodimers to bind to target sequences and strongly inhibits the transactivation activity of E2A-PBX1 [t(1;19) translocation, which occurs in 25% of pediatric pre-B cell acute lymphocytic leukaemia] (Abramovich C. et al JBC, 2000; Oncogene, 2002). It is highly expressed in human CD34+ progenitor cells, but is silenced in differentiated cells. To gain further insights into the possible functional role of HPIP and its domains and its possible role in a common pathway with HOX transcription co-factor PBX1, HPIP cDNA was cloned in pMSCV-IRES-YFP cassette. Umbilical cord blood enriched with CD34+ population of stem cells was obtained to perform in vitro and in vivo experiments. Mutants, with deletions of the microtubule binding region (ΔMBR-HPIP), and nuclear receptor and PBX1 interacting motif (ΔNRPID-HPIP) were generated and tested in vitro and in vivo. The constitutive expression of HPIP wt and ΔMBR-HPIP in human cord blood cells (CD34+) enhanced erythroid colony formation in CFC assay (p=0.008, n=6) while the ΔNRPID-HPIP mutant nullified the effect. Both mutants of HPIP augmented significantly, the formation of primitive colonies (GEMM and GM) in methylcellulose assay (p≤0.01, n=6) as compared to YFP control and HPIP wt. In replating CFC assays ΔNRPID-HPIP showed an increased number of myeloid colonies (p≤0.01, n=6) and GM (p=ns) colonies but a decrease in granulocytic colonies (p≤0.05, n=6) compared to YFP control and HPIP wt
Identification of Gfi1 as a Direct Target of the Leukemogenic Fusion Protein AML1-ETO9a
Authors: Lo Miao-Chia , Peterson Luke F. , Chen I-Ming , Willman Cheryl L. , Zhang Dong-Er
Published: April 2020

AML1-ETO9a (AE9a), a C-terminal splice variant of t(8;21), is highly leukemogenic in mice. Using combined gene expression and promoter occupancy (ChIP-on-chip) profiling of an AE9a induced murine AML model, we identified Gfi1, a transcriptional repressor important for neutrophil differentiation, as a direct target of AE9a. The level of Gfi1 mRNA was down-regulated following leukemia development. We further show that t(8;21) AML-M2 patient samples have lower Gfi1 mRNA levels compared to non-t(8;21) AML-M2 samples. In addition, Gfi1 protein was down-regulated in the human hematopoietic cell line U937 upon AE or AE9a expression. Two consensus AML1 sites (TGTGGT) located at positions −1750 and +405 from the Gfi1 transcriptional initiation site, respectively, are found in the proximity of the peaks of ChIP-on-chip profiling. We confirmed direct DNA binding of AE9a to these regions by ChIP assays. Furthermore, we showed that the activity of the Gfi1 promoter region −1880 to +481 is repressed by AE and AE9a in luciferase reporter assays. To address the effects of down-regulation of Gfi1 on proliferation, we overexpressed it in a primary AML1-ETO C-terminal mutant (AML1-ETOtr) leukemia cell line. Its overexpression slowed down growth and promoted apoptosis and differentiation of this cell line. In this report, we provide evidence that AE9a directly down-regulates Gfi1, thereby inhibiting hematopoietic differentiation, which may contribute to leukemia development.
Probing the Mitotic History and Developmental Stage of Hematopoietic Cells Using Single Telomere Length Analysis (STELA)
Authors: Hills Mark , Lucke Kai , Eaves Connie J. , Lansdorp Peter M.
Published: April 2020

In most somatic cells, telomeres shorten with each round of cell division. As a result telomere length can be used to assess the mitotic history of cells with some important caveats: the telomere length at birth is highly variable (presumably reflecting different alleles of genes regulating telomere length in the germline), telomere losses can be compensated by telomerase and the overall decline in telomere length includes sporadic, variable losses of telomere repeats resulting from damage to telomeric DNA and/or replication errors. The first caveat can be circumvented by testing cells from the same individual and sporadic telomere losses can be analyzed by single telomere length analysis (STELA). It is more difficult to exclude the effect of telomerase on telomere length but we have previously shown that, despite readily detectable expression of telomerase, hematopoietic stem and progenitor cells show a progressive decline in telomere length with cell division and with age. The clinical relevance of telomere shortening is illustrated in several recent studies linking very short telomeres to bone marrow failure and pulmonary fibrosis. We now show that purified human hematopoietic populations from mobilized peripheral blood (MPB) and cord blood (CB) enriched for stem cells (Lin−CD34+CD38−Rho−) and successively more mature cells display progressively shorter telomeres, pointing to the utility of this method for studies of the mitotic relationship between various stem and progenitor cells. Ultra-short telomeres were readily observed (and found to be significantly more frequent) in terminally differentiated cell populations of MPB, suggesting that sporadic telomere losses occur more frequently during differentiation. When 1000 Lin−CD34+CD38−Rho− cord blood cells were transplanted into two immuno-deficient mice, the most primitive human hematopoietic cells with a CD34+CD38− phenotype lost 3970 and 2790 bp respectively following regeneration in vivo, indicative of ~ 30–80 cell divisions assuming a telomere loss of 50–100 bp/division. Further losses in more differentiated cells were similar to those observed in cells before transplantation. These results illustrate the power of STELA for analysis of telomeres in rare cells and point to a novel strategy to study the turnover and replicative history of cells. Furthermore, these data demonstrate that self-renewal divisions in stem cells rather than additional cell divisions in downstream progenitors are the primary cause of telomere loss following transplantation.
The Dual Bromodomain Protein Brd2 Controls Primary B Cell Mitogenesis and Cell Cycle in Mice Reconstituted with Lentivirus-Transduced HSCs
Authors: Blanton Wanda P. , Wang Fangnian , Liu Hongsheng , Romesser Paul , Faller Douglas , Denis Gerald
Published: April 2020

Transcriptional control of cellular proliferation and differentiation is critically important in hematopoiesis; specifically, the role of chromatin-dependent regulatory processes in this context is poorly understood. The human BRD2 proto-oncogene encodes a double bromodomain protein that binds to acetylated histone H4 in chromatin and is located within the MHC class II locus, suggesting Brd2 plays a role in immunity. However, BRD2 shares no sequence similarity with other MHC genes, nor is Brd2 involved in antigen processing, but rather it plays a role in mitogenic signal transduction. We have previously found that whole-body knockout of Brd2 is lethal to mice. However, when Brd2 was expressed constitutively in the B cells of transgenic mice, Brd2 binds E2F proteins, histone acetylases and Swi/Snf complexes, and co-activates cyclin A leading to B cell lymphoma and leukemia. Importantly, elevated levels of Brd2 have been reported in primary malignant B cells from human and mouse. We therefore hypothesize that Brd2 multiprotein complexes, working through chromatin modification, are crucial in the control of the cell cycle and in the mitogen responsiveness and proliferation of the B cell compartment. To study the effects of Brd2 in B cell development and proliferation, we performed bone marrow transplants of hematopoietic stem cells in a chimeric mouse model. Hematopoietic stem cells were sorted from CD45.1 donor mice with the characteristic ‘side population’ profile by flow cytometry and transduced with lentivirus containing vectors for Brd2 overexpression, shRNA knockdown, or control vectors. Recipient CD45.2 mice were lethally irradiated and a functional immune system was successfully reconstituted with donor cells and CD45.2 competitor BM cells. Mice were immunophenotyped and functional B cell mitogenic capacity was examined by BrdU incorporation into LPS-stimulated B cells. We found that in the spleen, Brd2 expression dramatically expands the CD45.1 (but not CD45.2) B cell compartment at the expense of T cells and renders B cells mitogenically hypersensitive. Compared with control, there was an increase in BrdU incorporation at 24 and 48 hours (29.8% v. 43.5% at T=24 h; 56.9% v. 66.7% at T=48 h). Preliminary results also suggest that B cell development was skewed in the bone marrow and periphery towards B1a phenotype. Moreover, downregulation of Brd2 via shRNA blocked cyclin A transcription and completely arrested B cell development and proliferation. Taken together, these data suggest that Brd2, through epigenetic regulation of the cell cycle, plays an important role in B-lymphopoiesis, proliferation, and stimulation.