bluuebunny/crossref_metadata_2025 huggingface dataset:
Prognostic Analysis of Pre-Transplant CD26-Positive Lymphocytes and CD26bright/CD45RO-Positive Memory T-Cell Levels in Patients Receiving an Autologous Hematopoietic Progenitor-Cell Transplant.
Authors: Hildebrandt Martin , Gollasch Hella , Rautenberg Kirstin , Doerken Bernd , Ludwig Wolf-Dieter
Published: April 2020

The lymphocyte surface glycoprotein CD26 is involved in an array of diverse signalling pathways and costimulatory events. CD26positive cells possess dipeptidylpeptidase IV (DPP IV) activity, a serine protease known to inactivate SDF-1/CXCL12, a key mediator of stem cell homing and engraftment. Furthermore, CD26 modulates surface expression of CTLA-4 and contributes to T-cell migration through endothelial layers. CD26 has been attributed with a central role in alloantigen-mediated immune pathways and memory T-cell responses. The purpose of this study was to evaluate the levels of a distinct memory T-cell subset, CD26bright/CD45RO-positive, in autologous hematopoietic progenitor-cell transplant (HPCT) recipients. Between 2003 and 2006 we enrolled 42 patients scheduled to undergo high-dose chemotherapy and autologous HPCT (multiple myeloma, n=31; Hodgkin’s Disease, n=3; NHL, n=6; PNET, n=1; AML, n=1). Levels of memory and naïve CD26, CD34, CD4, CD8, as well as co-expression of CD4 or CD8 among CD26bright/CD45RO-positive cells were analyzed before autologous HPCT. In addition, the number of memory and naïve CD26-positive T cells transfused per kg body weight in the progenitor cell harvest were determined. The subsets of CD26-positive cells were correlated with kinetics of engraftment and with the occurrence of disease progression or relapse after autologous transplantation. With regard to kinetics of engraftment, only the number of CD34-positive cells transfused was associated with rapid engraftment (P=0.001). However, CD26-positive cells were of predicitve value for the occurrence of disease progression or relapse. Pre-transplant CD26-positive T-cell levels correlated with progression-free survival (PFS) (P=0.022). Specifically, the number of CD26bright/CD45RO-positive memory T-cells in the autograft correlated with PFS and, in regression analyses, emerged as the only variable predictive for the occurrence of diease progression or relapse (P=0.006). The prognostic effects of pre-transplant CD26bright/CD45RO-positive memory T-cells were independent of the type of disease and of the conditioning regimen applied. The analysis of antigens CD4 and CD8, respecively, on CD26bright/CD45RO-positive T-cells yielded no additional information. Our results suggest that pre-transplant levels of CD26-positive T cells, specifically a memory cell subset of CD26bright cells coexpressing CD45RO, may yield information with regard to outcome in autologous HPCT recipients. These observations may contribute to a prospective identification of those patients at higher risk of relapse, based on their immune status.
Avoidance of Post-Thaw Wash Prior to Transplantation of Plasma Depleted Cord Blood (PD CB) Is Associated with Improved Engraftment & Decreased Severity of Chronic GvHD (cGvHD) without Increased Relapse.
Authors: Chow Robert , Wang Brian , Rosenthal Joseph , Nademanee Auayporn , Karanes Chatchada , Jaing Tang-Her , Graham Michael , Delaney Colleen , Gjertson David , Petz Lawrence , Forman Stephen , Kurtzberg Joanne
Published: April 2020

Post-thaw washing of frozen CB prior to transplantation is widely practiced to remove DMSO, red cell debris and free hemoglobin, however, washing a red cell containing product may result in cell loss. We analyzed outcomes of engraftment, survival, relapse and GvHD in 299 patients transplanted with plasma depleted but not red cell reduced (PD) CB where 154 were washed (W) and 145 were directly infused - (non-washed or NW). 207 (69%) of 299 patients had malignant diagnosis. The two groups were comparable for patient, disease and graft characteristics except for a higher % of Asian patients in the NW group in contrast to a higher % of Caucasian/Hispanic patients, higher % of US transplant centers (TC) and higher number of malignant diagnoses for the W group. Nucleated cell recovery after thawing, as reported by TC, was higher for NW (median 85% vs. 79%, p=0.003). The NW group had higher cumulative incidences of and faster neutrophil (ANC500) and platelet engraftment (Plt 20K and 50K) than the W group. No difference was seen for acute GvHD (aGvHD); however, the NW group had significantly lower rates of extensive (Ext) cGvHD and higher rates of limited (Ltd) cGvHD (p=0.0005 and <0.0001). Relapse rates were similar between W and NW. Multivariate analysis and matched pair analysis between W versus NW confirm these observations. In univariate analysis, overall survival (OS) and disease-free survival (DFS) were significantly higher for the NW group. This retrospective analysis represents the largest series comparing different thaw methods for PD CB and is the first to demonstrate a potential association of no post-thaw wash with significant decrease in extensive cGvHD rate without concomitant increase in relapse rate. These improvements may contribute to the apparently better survival for the NW group. Prospective trials which may include infusion of wash supernatants into patients are necessary to confirm and better understand these observations.

ANC 500 & Plt 20K 100 Day TRM 1 & 3 Yr OS 1 & 3 Yr DFS 1 Yr Relapse II-IV/III-IV aGvHD Ltd/Ext cGvHD All results in % except for p-values & where indicated. Engraftment presented as cumulative incidence, GvHD as proportions; all others estimated by Kaplan-Meier. Overall 88±4/69±4 17±2 61±3 56±3 51±3 39±5 23±3 35/14 20/11 W 87±5/64±5 17±3 54±4 48±4 43±5 30±6 27±5 36/15 9/17 NW 91±6/76±6 16±3 69±4 67±4 60±5 57±5 20±4 36/14 36/1 P value 0.002/0.001 0.05 0.02 0.05 0.29 0.93/0.80 <0.0001/0.0005 ANC 500 & Plt 20K TRM OS DFS Relapse II-IV/III-IV aGvHD Ltd/Ext cGvHD MV adjusted Cox RR & P value 1.51 0.002/1.70 0.001 0.67 0.11 0.71 0.09 0.74 0.11 0.76 0.34 N/A 3.88/<0.001 Matched Pairs P value 0.008/0.0002 0.53 0.32 0.22 0.19 0.98/0.44 0.02/0.001
Puberty, Gonadal Function and Pregnancy in Female Patients Following Hematopoietic Cell Transplantation (HCT).
Authors: Sanders Jean E. , Flowers Mary E.D. , Carpenter Paul C.
Published: April 2020

With increasing success of myeloablative HCT for patients with hematologic disorders, development of pre-pubertal children through puberty, return of gonadal function and the ability to become pregnant are of concern. To determine the impact of the preparative regimen on these issues, long-term follow-up records of more than 2,000 patients have been evaluated. Preparative regimens include cyclophosphamide (CY) 200 mg/kg, busulfan (BU) 16 mg/kg plus 120 or 200 mg/kg CY, or CY 120 mg/kg and 12–15.75 Gy total body irradiation (TBI). Among girls who were pre-pubertal at HCT and are now >12 years of age, 24/26 (92%) CY recipients, 11/23 (52%) BUCY recipients, and 46/96 (51%) CY + TBI recipients have developed normally through puberty. Among 148 post-pubertal women transplanted with CY only, 75 (50%) recovered gonadal function at a median of 6 months after HCT and 45 (30%) have become pregnant. This is in contrast to 2 (1%) of the 207 women who received BUCY who have shown gonadal function recovery and 1 has become pregnant. Among the fractionated TBI recipients, 39 (8%) of 460 women who received CY + 12 Gy TBI recovered ovarian function and 17 (3%) have become pregnant whereas among the 366 women who received 14.0–15.75 Gy TBI, 14 (4%) have recovered ovarian function and 4 (1%) have become pregnant. All women who recovered ovarian function after BUCY or TBI regimens were <25 years of age at HCT. The primary pregnancy complication was preterm labor and delivery, but all babies born were normal. For those women who do not recovery ovarian function, in vitro fertilization may be an option, and 14 women in this category have had successful pregnancies with 21 babies born using donated oocytes or pre-HCT stored gametes. These data demonstrate that pre-pubertal girls have more than 50% rate of developing normally through puberty and among women <25 years of age at HCT, recovery of ovarian function varies between 1%-50% depending on preparative regimen and some have become pregnant. For those who do not recover ovarian function, in vitro fertilization may be an option.
The Degree of Telomere Loss in Hematopoietic Cells Correlates with the Risk of Secondary Myelodysplasia/Acute Leukemia Development Following Autologous Stem Cell Transplantation.
Authors: Ricca Irene , Rocci Alberto , Ruella Marco , Zanni Manuela , Caracciolo Daniele , Ladetto Marco , Boccadoro Mario , Ferrero Dario , Tarella Corrado
Published: April 2020

Introduction: Secondary myelodysplastic syndrome and acute myelogenous leukemia (sMDS/AML) may occur following autologous stem cell transplantation (ASCT). The molecular pathogenesis of sMDS/AML is uncertain; moreover, no suitable indicators able to define the risk of sMDS/AML development have been identified so far. A marked though variable telomere loss has been observed in hematopoietic cells following ASCT; meanwhile, shortening of telomere length has been observed to be associated with several neoplasias, including haematological malignancies. Thus, telomere dynamics has been investigated in a series of patients who received ASCT, in order to verify whether the degree of telomere loss in hematopoietic cells might be associated with the risk of sMDS/AML development.

Methods: Telomere length (TL) was retrospectively evaluated in bone marrow (BM) cells from 38 lymphoma patients (M/F=24/14; median age=51 years, range 24–68) long-term survivors following ASCT and from 51 healthy donors (M/F=31/20; median age=53 years, range 18–82). Median follow-up since ASCT was 6 years (range 1–10). All patients were in continuous complete remission and displayed normal haematological values at the time of TL assessment. Among 38 autografted patients, 7 developed sMDS/AML (3 AML, 3 sMDS, 1 persistent pancitopenia with cytogenetic abnormalities), at a median of 5 years (range 1–10) following transplant. There were no significant differences in terms of demographical and clinical features not even for the amount of CD34+ve cells reinfused (median values: 5.2 vs 6.8 × 106 CD34+ cells/kg, respectively), between patients developing sMDS/AML and the remaining autografted patients. Samples for TL analysis were obtained and stored at a median of 12 months (range 6–24) before clinical development of sMDS/AML. TL was evaluated by Southern blot analysis.

Results: TL of autografted patients was found to be significantly shorter compared to that of age-matched healthy donors, consistently with previous reports (see Figure 1, squares=healthy controls, circles=autografted subjects). A further TL loss was observed in all the 7 patients who subsequently developed sMDS/AML; their TL (Figure 1, black circles) was significantly shorter compared to both healthy subjects and sMDS/AML-free autografted patients (Figure 1, grey circles) (p<0.0005 and p<0.01, respectively).

Conclusions. A marked telomere loss seems to be a predictive marker of the development of sMDS/AML following ASCT; TL analysis can now be considered among follow-up assays, in order to early identify patients at high-risk of sMDS/AML occurrence following autograft.

Figure 1. TL of BM mononuclear cells in autografted patients vs healthy subjects Figure 1. TL of BM mononuclear cells in autografted patients vs healthy subjects
Ataxia Telangiectasia Mutated (ATM) Gene Expression Is Reversibly Up-Regulated in FANCA-Mutant Patient Fibroblasts by an Indirect Mechanism.
Authors: Liu Johnson M. , Nihrane Abdallah , Yamamoto Kazuhiko
Published: April 2020

Fanconi anemia (FA) predisposes to hematopoietic failure, birth defects and cancer, particularly leukemia and squamous cell carcinomas involving the cervix or head and neck. Although the FANC proteins have been shown to be involved in DNA repair, the pathogenesis of bone marrow failure and other developmental abnormalities is still unclear, but presumably, reflects abnormalities in senescence and apoptosis. The canonical ATM-p53 pathway is a pivotal mediator of the DNA damage response, which may act as a barrier to cancer through cell cycle and apoptosis regulation but has not been fully studied in FA. To address this, we first analyzed the integrity of the ATM-p53 axis in primary fibroblasts derived from FA patients. By immunoblot assays, we found that expression and phosphorylation of ATM and p53 were all up-regulated in two different FANCA-mutant fibroblasts following irradiation (IR) or mitomycin C (MMC) treatment, when compared to identically treated isogenic mutant cells transduced with wild-type FANCA cDNA. We reasoned that these results might be explained by up-regulation of the basal levels of ATM protein in the mutant fibroblasts. By real-time PCR, we confirmed that ATM gene expression was increased in the mutant line in comparison to the gene-corrected control. We also confirmed the up-regulation of both ATM protein and transcription in a FANCA-mutant EBV-immortalized lymphoblastoid cell line in comparison to its gene-corrected control. In order to determine if these changes in ATM gene expression were directly caused by FANCA depletion, we transfected HCT116 cells (known to be p53 wild-type) with two different siRNAs against FANCA. For these experiments, we used a plasmid expression vector (OriGene Technologies, Rockville MD) that drives 29-mer short hairpin RNAs targeting FANCA. In both transient and stable transfection experiments with either siRNA, we found that knockdown of FANCA expression was associated with down-regulation of ATM expression, as assessed by real-time PCR, when compared to cells transfected with an empty vector control. These results have led us to hypothesize that knockdown of FANCA leads to down-regulation of ATM, whereas cells with mutations in FANCA up-regulate ATM, perhaps as a result of chronic genotoxic stress not seen in the knockdown cells. Up-regulation of the ATM-p53 axis, in turn, may contribute to the hypo-proliferative characteristics seen in both primary fibroblasts and hematopoietic stem cells from FA patients.
Fanconi-Like Syndrome with Negative Chromosomal Breakages Test and High Incidence of Myelodysplasia.
Authors: Gupta Sunil , Rassam Saad M.B. , Riyali Madan , Ho Aloysius Y.L. , Mufti Ghulam
Published: April 2020

Fanconi’s anaemia (FA) is a syndrome of defective chromosomal repair characterised by bone marrow failure, skeletal and visceral abnormalities, progression to myelodysplasia (MDS) and acute myeloid leukaemia (AML), hypersensitivity to alkylating agents and increased chromosomal breakage using diepoxybutane (DEB) testing. We describe 13 patients who presented with FA like clinical and/or haematological features who had negative DEB testing. There were 9 males and 4 females with an average age of presentation of 23 years (range 1.5 months - 53 years). They had isolated neutopenia (1/13) or anaemia (1/13), bicytopenia (4/13) or pancytopenia (7/13) at first presentation. Haemoglobin level ranged between 3.8 - 15 g/dl (mean 9.7), white cell count 0.64 - 8.4 × 109/l (mean 2.88), neutrophils 0.05 - 5.43 × 109/l (mean 1.14) and platelets 30 - 179 ×109/l (mean 82). Five patients had macrocytosis with mean cell volume (MCV) above 100fl. The most common morphological features noticed were high arched palate, short stature and short fingers and toes. Seven patients had hypocellular marrow with trilineage dysplasia (refractory anaemia), one had hypocellular marrow with refractory anaemia with ring sideroblasts, 3 patients had hypocellular marrow and AML and 2 had hypocellular marrow with no morphological dysplasia. DEB testing for increased chromosomal breaks was negative in all the 13 patients. Nine patients had cytogenetic abnormalities with monosomy 7 or complex abnormalities being the most common. Three patients have stable blood counts on a watch and wait policy 6–24 months from diagnosis. Five patients’ cytopenias progressed responding to treatment with androgens (2) and anti-thymocyte globulin with steroids (1) and 2 required allogeneic stem cell transplantation (BMT). Androgen responses were sustained in one patient for over 12 years and was followed by AML transformation in the other. Two patients are lost to follow up, 1 died following BMT for AML and 2 had severe mucositis with AML therapy fatal in one and associated with prolonged pancytopenia in another. Four patients had BMT, 2 are in remission, 1 died post BMT and 1 failed. FA phenotype with cytopenia and subsequent progression to MDS/AML is well known and diagnosis is usually confirmed with DEB testing. It is important, however, to recognise FA like syndromes with negative DEB, who appear to have similar clinical behaviour and sensitivity to chemotherapy, as therapy may differ from standard cases of aplastic anaemia, MDS or AML. Genetic testing for the described Fanconi genes may shed more light into those cases.
Cell Therapy in a Mouse Model of Immune-Mediated Bone Marrow Failure.
Authors: Chen Jichun , Young Neal S.
Published: April 2020

Immune-mediated bone marrow (BM) failure has been modeled in the mouse by infusion of lymph node cells from allogeneic C57BL/6 (B6) donors into major or minor histocompatibility antigen-mismatched recipients (Chen et al., Blood 2004; Bloom et al., Exp Hematol 2004, Chen et al., J Immunol 2007). Co-infusion of limited numbers of CD4+CD25+ regulatory T lymphocytes (Tregs) can alleviate clinical manifestations by suppressing the expansion of pathogenic T cells (Chen et al., J Immunol 2007). In the current study, we investigated the effectiveness of Tregs and suppressor cells contained in BM stroma in this fatal disease. Infusion of fewer than 3 × 103 Tregs to each recipient mouse had only a minor effect in preserving BM cells and did not prevent pancytopenia. Fifteen-50 × 103 thymic Tregs was moderately protective: blood WBC, RBC, platelet and BM cell counts at three weeks after cell infusion were 197%, 116%, 155% and 158% of those of control animals that did not receive Treg infusion; 5–10 × 103 B6 splenic Tregs produced the largest effect as WBC, RBC, platelet and BM cell counts were 275%, 143%, 276%, and 198% of controls. Overall, Treg therapy was helpful but its effectiveness was limited and variable among individual recipients as no antigen-specific Tregs can be identified for the treatment of BM failure. Learned about the immunosuppressive effects of mesenchymal stem cells (MSCs), we went on to test the effectiveness of stromal cells as another therapeutic modality for BM failure, since stromal cells contain MSCs. These cells were derived from B6 BM by culture in α-modified Eagle medium at 33°C with 5% CO2 for two weeks. After separating the non-adherent cells, we detached the adherent stromal cells and infused them into TBI + B6 LN-infused C.B10 mice. Injection of 106 stromal cells at the time of LN cell infusion effectively preserved WBCs (3.09 ± 0.51 vs 0.61 ± 0.18), RBCs (8.72 ± 0.14 vs 3.52 ± 0.46), platelets (924 ± 93 vs 147 ± 25) and BM cells (186.6 ± 8.7 vs 52.7 ± 7.8) when compared to LN-cell-infused mice without stromal cell addition. Delayed stromal cell injection at day 9 after LN cell infusion had only a mild effect on the preservation of RBCs (147%), platelets (276%) and BM cells (223%) and no effect on WBCs (64%), and infusion of non-adherent cells from the same stromal cell culture had no therapeutic effect. Stromal cell-infused mice had higher proportion of FoxP3+CD4+ cells in the peripheral blood (59.7 ± 10.7% vs 29.8 ± 5.4%) and more Lin−CD117+CD34− hematopoietic stem and progenitor cells in the BM (591 ± 95 vs 60 ± 43, thousand) in comparison to LN cell infused mice without stromal cell treatment. Mitigation of pathogenic T cells, including both CD4 and CD8 T lymphocytes, is the potential mechanism for the effectiveness of Treg and stromal cell therapies that helped to protect hematopoietic stem and progenitor cells in the BM of affected animals.

Figure Figure
Marked Improvement in Short- and Long-Term Survival in Severe Aplastic Anemia Patients Treated with Immunosuppression in the Past 18 Years.
Authors: Scheinberg Phillip , Wu Colin O. , Nunez Olga , Young Neal S.
Published: April 2020

Forty years ago severe aplastic anemia (SAA) was almost universally fatal disease, but now most patients can be successfully treated with hematopoietic stem cell transplantation (HSCT) or immunosuppressive therapy (IST). Since the majority of patients are not suitable candidates for allogeneic HSCT from a histocompatible sibling, IST is usually the treatment of choice. From the introduction of the standard anti-thymocyte globulin (ATG) + cyclosporine (CsA) regimen in the 1980s, the hematologic response rate has been 60–70% despite efforts to improve on the ATG+CsA platform. Across many protocols, there is a strong link between response to IST and survival. We examined short and long term survival of patients who were treated with initial IST at the Clinical Center of the National Institutes of Health from 1989 to 2006; a total of 372 patients were analyzed in 3 separate cohorts: those who received IST from 1989–1995; 1996–2001 and 2002–2006. Thirty-two deaths occurred within the 6-months period of IST with 15 being attributed to a fungal infection. The distribution of early deaths (< 6 months) in relation to the different time periods were 15% (1989–1995); 9% (1996–2001); and 5% (2002–2006) (p = 0.003). Survival beyond 6 months also improved (1989–1995 and 2002–2006, 65% vs. 85%, respectively; p < 0.01). Surprisingly, the survival benefit was most striking among patients who did not respond to IST at 6 months. Among the total 116 non-responders, the number of patients who received a second course of IST was 0% (1989–1995), 37% (1996–2001) and 51% (2002–2006); those who underwent HSCT were 19% (1989–1995), 23% (1996–2001) and 18% (2002–2006); and those who did not receive a second course of IST or HSCT were 83% (1989–1995), 29% (1996–2001) and 11% (2002–2006). We draw two major conclusions from our data analysis. First, improvement in short term survival over the last two decades is most likely due to advances in supportive care, as the response rate to horse ATG + CsA has remained largely unchanged during the same time period. The marked decline in deaths due to invasive Aspergillosis infection suggests that the introduction of effective and relatively nontoxic antifungal drugs is of prime importance (e.g., voriconazole in 2002 and caspofungin in 2004). Second, the utilization of secondary therapies--a repeat course of IST and allogeneic HSCT for older adults and matched unrelated transplants for younger individuals--have salvaged patients who are primary ATG-failures. The advances in supportive care also impact long term survival in those who receive salvage therapies and in those who fail repeated courses of IST and are not suitable candidates for HSCT, who now can be adequately maintained on growth factor and transfusion support, often for years. Current survival rates in SAA should be weighed when in considering treatment algorithms for patients who fail initial IST.
Identification of Alternative Splicing Changes in Erythroid Differentiation Using Human Exon Arrays.
Authors: Yamamoto Miki L. , Kang Jeong-Ah , Arribere Josh A. , Wickrema Amittha , Conboy John G.
Published: April 2020

Terminal erythroid differentiation is accompanied by extensive structural remodeling as the cell enucleates and eventually assumes the biconcave disk morphology of the mature cell. Previous studies have documented many changes at the transcriptional level essential for erythroid differentiation. Changes in erythroid gene expression also occur at the level of pre-mRNA alternative splicing: the activation of 4.1R (EPB41) exon 16 splicing in late erythroblasts increases 4.1R affinity for spectrin-actin and mechanically strengthens the plasma membrane. We hypothesize that analogous changes in alternative splicing affect the structure and function of other erythroid proteins. To identify additional alternative splicing switches in erythroid genes, a genome-wide exon expression analysis was carried out using the new Affymetrix Human Exon 1.0 ST Array. Unlike traditional gene expression microarrays, this array has single exon resolution and can detect changes in expression due to alternative splicing. Samples for array analyses were prepared from RNA of human erythroid progenitor cells grown in culture for 7, 10, and 14 days, corresponding to basophilic, polychromatic, and orthochromatic stages. Analysis of this exon array data confirmed that 4.1R exon 16 splicing was activated in day 14 cells, and that a known inhibitor of exon 16 splicing, hnRNP A1, was down-regulated in coordination with the 4.1R splicing switch. As another positive control, we confirmed in array data the expression of a known erythroid-specific 3′ end in beta-spectrin mRNA in all three time points of erythroblasts, while array data from muscle tissue showed expression of only the non-erythroid 3′ end of beta-spectrin. Array data is now being analyzed to identify new cases of alternative splicing during erythropoiesis, and confirmation of several candidate splicing switches by RT-PCR and quantitative PCR is under way. A number of genes, including PIK3R1, SLC12A6, and TNPO2, show changes in alternative 5′ first exon usage during late erythropoiesis. A splicing change involving an internal cassette exon in MBNL2, which encodes a splicing regulator, was identified by array data and confirmed by RT-PCR. In addition, overall gene expression analyses confirm up-regulation of known genes expressed during erythroid differentiation, including Band 3, GLUT1, ALAS2, and BCL2L1. This preliminary analysis demonstrates the application of exon arrays toward the identification of splicing switches that occur during differentiation of human erythroblasts. Further validation of putative alternative splicing events is in progress, and investigation of the regulation of the validated events and the physiological implications of the predicted changes in the proteins will be pursued in the future.
High Resolution HLA Matched Unrelated Donor Versus HLA Identical Sibling Transplantation for Chronic Phase CML.
Authors: Arora Mukta , Spellman Stephen , Haagenson Michael , Klein John P. , Hurley Carolyn , Selby George , Antin Joseph , Kernan Nancy , Kollman Craig , Nademanee Auayporn , Weisdorf Daniel J. , Horowitz Mary M. , McGlave Philip , Petersdorf Effie
Published: April 2020

Current criteria for the selection of adult volunteer unrelated donors (URD) include matching for HLA alleles determined by high resolution typing methods. However, the impact of URD matching for 8/8 HLA alleles (HLA-A, B, C, DRB1) on survival compared to HLA identical sibling donor bone marrow transplant (BMT) remains undefined. We tested this hypothesis in a homogenous population of patients with good-risk chronic myeloid leukemia (CML) in first chronic phase (CP1) where a strong allogeneic effect and hence a lower risk of relapse is anticipated. In total, 939 URD bone marrow transplant recipients with CML in CP1 were retrospectively typed for HLA -A, B, C, DRB1, DQ and DP alleles. Outcomes were compared to 3514 HLA identical sibling donor bone marrow transplant recipients (CML, CP1). Five-year survival after 8/8 matched URD HCT was slightly, but significantly worse than that after sibling BMT (54% versus 63%). [RR 1.36 (95% CI 1.17–1.59), p=0.0001]. In multivariate analysis (model adjusted for time from diagnosis to transplant, CMV serostatus of donor and recipient, year of transplant, and T-depletion, and stratified on donor and recipient gender mismatch and radiation use), a single allele mismatch at any class I locus or at HLA-DRB1 led to significantly poorer survival. Risks for single class I versus single DRB1 mismatches were similar. Two locus mismatching was associated with higher risk compared to matched or single locus mismatching. In this homogenous cohort of good risk patients with CML in CP1, 5 year survival after transplant from 8/8 allele-matched donors begins to approach the results of sibling donor transplantation. Additive effects of multi-locus mismatching are not well tolerated and should be avoided if possible.

N Relative risk (95% CI) P-value 5 year survival * Antigen or allele mismatch, **allele mismatch HLA identical siblings 3514 1.00 63% (61–64%) 8/8 matched 531 1.36 (1.17–1.59) 0.0001 54% (50–58%) 7/8 A mismatch* 68 1.90 (1.36–2.65) 0.0002 43% (32–55%) 7/8 B mismatch* 34 2.77 (1.81–4.23) <0.0001 30% (15–48%) 7/8 C mismatch* 113 1.88 (1.45–2.43) <0.0001 42% (33–51%) 7/8 DRB1 mismatch* 37 2.07 (1.37–3.12) 0.0005 37% (23–54%) 6/8 Class I mismatches* 128 2.66 (2.11–3.35) <0.0001 33% (25–42%) 6/8 Class I mismatch* and DRB1 mismatch** 28 3.40 (2.23–5.19) <0.0001 21% (8–37%) 7/8 DRB1 mismatch** vs. 7/8 Class I mismatch* 37/215 1.03 (0.67–1.61) 0.88N/A
Functional Polymorphisms of Notch 4 and the Risk of Aplastic Anemia.
Authors: Kim Hee Nam , Kim Yeo-Kyeoung , Lee II-Kwon , Yang Deok-Hwan , Shin Myung Geun , Kook Hoon , Hwang Tai Ju , Park Moo Rim , Jo Deog Yeon , Won Jong Ho , Kim HyeoungJoon
Published: April 2020

Aplastic anemia (AA) is a heterogeneous disease characterized by bone marrow aplasia and peripheral blood pancytopenia, a profound deficit of hematopoietic stem and progenitor cells. Notch4 is structurally distinct from Notch1 through Notch3. Notch4 signaling plays a role in lineage commitment in hematopoiesis. Therefore, we hypothesized that Notch4 polymorphisms could potentially influence the expression level of Notch4 protein and associate with the risk of AA. To investigate the role of the two Notch4 rs2071282 (+2763 C>T, Pro204Leu) and 422951(+3323 A>G, Thr320Ala) polymorphisms in susceptibility to AA, we genotyped in 151 AA patients and 552 healthy control subjects using PCR-RFLP and quantified expression of Notch4. We previously reported that Notch4 +2763 CT genotype is significantly associated with increased risk for AA (odds ratios (OR) = 1.77; 95% confidence intervals (CI) = 1.05–2.98, p=0.032) and the haplotype CG is significantly associated with the risk of AA (OR= 0.70 95% CI 0.50–0.98, p= 0.037) (The American Society of Hematology 2006. 12. poster). With quantified expression level of Notch4 using quantitative SYBR green methods, we identified a significant positive correlation between +2763 C>T and +3323 A>G each genotype and transcription level (p<0.0001) in AA. With quantified expression level of Jagged 1, we also identified a significant positive correlation between +2763 C>T and +3323 A>G each genotype and transcription level (p<0.0001) in AA. To the effect of the SNP on transcription, we also carried out allelic imbalance using allelic quantification mode pyrosequencing method. The mean ratio was 1.7, higher than that of controls cDNA and DNA amplicons of AA (ratio=1.3, P < 0.001) in the 16 heterozygotes AA and 25 contrrols with genotype +3323 A/G, but there was no significance correlation in heterozygotes with genotype +2763 C>T. In conclusion, our results suggest that regulation of expression Notch4 by two polymorphisms may play an important role in the susceptibility to AA.
Inhibition of Factor VIIa-Tissue Factor by Protein C Inhibitor.
Authors: Fortenberry Yolanda , Cook Anne B. , Church Frank C.
Published: April 2020

Protein C inhibitor (PCI) is a plasma serine protease inhibitor (serpin) that inhibits several proteases in blood coagulation including thrombin, thrombin-thromobomodulin and activated protein C. Although PCI has been shown to inhibit these key blood coagulation proteases, a clearly defined hemostatic role for PCI remains to be elucidated. Preliminary results from our laboratory show that exogenously added PCI prolongs venous thrombus formation upon blood vessel injury in a murine model of vascular injury. These results suggest that PCI may be responsible for inhibiting other proteases in blood coagulation, possibly including factor VIIa. Blood coagulation is initiated when factor VIIa binds to TF on cell surfaces. We investigated the ability of PCI to inhibit factor VIIa, both in the presence and absence of TF in vitro, and in a cellular environment. It has been shown previously and we have confirmed that the serpin antithrombin (AT) inhibits both factor VIIa and factor VIIa/TF; however, the ability of PCI to inhibit this protease has not been fully investigated. PCI and AT inhibit factor VIIa in the presence of TF and heparin (AT: k2=1.1 × 104 M−1 s−1; PCI: k2=7.2 × 103 M−1s−1). In the absence of heparin the inhibition of factor VIIa/TF by PCI is reduced 10-fold. Interestingly, PCI is unable to inhibit VIIa in the absence of TF with or without heparin. Furthermore, both PCI and AT form SDS-stable complexes with factor VIIa (in the presence of TF). We also investigated whether PCI is able to inhibit VIIa-mediated cell migration of the breast cancer cell line MDA-MB-231 that constitutively expresses TF. AT and PCI are able to inhibit cell surface derived factor VIIa/TF. Interestingly, the rate of factor VIIa inhibition on MDA-MB-231 cells is 1.2 and 0.4 × 103 M−1s−1 for PCI and AT, respectively. The factor VIIa-mediated cell migration of MD-MB-231 cells is attenuated in the presence of PCI, but not in the presence of the PCI inactive mutant (T341R). Given that both AT and PCI are able to inhibit factor VIIa/TF both in vitro and in a cellular environment, suggests that more potent derivatives (prepared by site-directed mutagenesis) of these serpins may be useful alternatives as novel therapeutics anticoagulants that target the initial step in the coagulation pathway.
The Effect of Aquaporin 2 Gene Variations on Plasma Levels of von Willebrand Factor and Factor VIII and on the Risk of Venous Thrombosis.
Authors: Nossent Anne Yael , Ellenbroek Johanne H. , Frolich Marijke , Rosendaal Frits R. , Bertina Rogier M. , Knoers Nine V.A.M. , Eikenboom Jeroen C.J.
Published: April 2020

High levels of von Willebrand factor (VWF) and factor VIII (FVIII) are a risk factor for thrombosis. Determinants of high VWF and FVIII levels are poorly understood. Secretion of VWF from endothelial storage pools is regulated by vasopressin type-2 receptor (V2R). Previously, we have shown that a V2R variant, which has increased binding affinity for its ligand vasopressin (AVP), is associated with increased levels of VWF and FVIII1. Nephrogenic Diabetes Insipidus (NDI) is a disorder characterized by renal insensitivity to AVP, caused by mutations in the genes encoding V2R or the aquaporin 2 water channel (AQP2). AQP2 expression is enhanced by stimulation of V2R. Patients with NDI are unable to concentrate their pre-urine. We hypothesized that carriers of AQP2 mutations compensate excess fluid loss by up-regulating AVP release and V2R expression, resulting in increased VWF and FVIII secretion. To test this hypothesis, we set up the Factor Eight in Nephrogenic Diabetus Insipidus study (FENDI), which includes 13 NDI families: 14 NDI patients (12 V2R- and 2 AQP2-linked), 14 carriers (9 V2R- and 5 AQP2-linked) and 25 unaffected family members, as well as 48 unrelated healthy individuals. In addition, we looked at effects of common AQP2 gene variations in a case-control study on venous thrombosis, the Leiden Thrombophilia Study (LETS), which consists of 474 patients with a first deep vein thrombosis and 474 healthy controls, sex and age matched to the patients. In the FENDI, no differences were observed between NDI patients, carriers and unaffected individuals in markers for fluid homeostasis such as hematocrite, serum osmolality and blood pressure. AVP reached detectable levels in all carriers of AQP2 mutations, compared to 27% and 56% in unrelated and related unaffected individuals, respectively. AVP levels, were, when detectable, elevated in all patients and carriers. VWF propeptide, a measure of the VWF secretion rate, VWF antigen and FVIII activity were also highest in carriers of AQP2 mutations. In the LETS, we sequenced a 6.6 kb long genomic region around the AQP2 gene in 25 selected individuals. We identified 18 single nucleotide polymorphisms (SNPs), of which 16 were genotyped in the entire LETS. Although reliable haplotypes could not be formed, due to recombination, the SNPs were linked within 5 clusters. In three of these clusters, up to 2.5-fold increases in thrombosis risk were observed. In these same clusters we observed associations of the AQP2 SNPs with arterial blood pressure. However, none of the AQP2 SNPs were associated with VWF or FVIII levels in healthy controls of the LETS. In conclusion, increased AVP levels in carriers of NDI-causing AQP2 mutations appear associated with increased VWF secretion. Furthermore, in the LETS, common AQP2 gene variations are associated with the risk of venous thrombosis.
LDL Receptor Mediates Effective Endocytosis of Activated Factor V.
Authors: Meijer Alexander B. , Roosendaal Sigrid D. , Limburg Vincent , van der Zwaan Carmen , Rodenburg Kees W. , Mertens Koen
Published: April 2020

The platelet α-granules contain a unique pool of partially activated factor V, which has been suggested to originate form megakaryocyte endocytosis of factor V (FV) from plasma. The presence of activated FV (FVa) in plasma itself should be tightly controlled as dysfunction therein may predispose to thrombotic disorders. Previously, we have reported that FVa but not FV can bind the low-density lipoprotein receptor related protein (LRP). This multifunctional receptor can bind a multitude of ligands including FV’s homologue factor VIII (FVIII). We now investigated whether FV or FVa can, like FVIII, also bind the more restricted LDL receptor (LDLR). To this end, the endocytosis of FV and FVa by CHO cells expressing LDLR (CHO-LDLR+ cells) was assessed utilizing confocal microscopy. In the experimental setup, FV and FVa were visualized employing immuno-fluorescence staining techniques. The results showed that within 10 minutes after addition of FVa, fluorescent spots appeared inside the CHO-LDLR+ cells. In contrast, no fluorescent spots were observed after 10 minutes of incubation with FV. These observations suggest that FVa but not FV effectively interacts with the LDLR expressing CHO cells. We then assessed whether FVa can compete with FVIII for endocytosis by CHO-LDLR+ cells. In the presence of an equimolar amount of FVa and a FVIII derivative (FVIIIYFP) containing yellow fluorescent protein, both proteins were detected within the same vesicles inside the CHO-LDLR+ cells. Employing co-localization studies, we established that these vesicles represented early endosomes. In the presence of an access of FVa, however, the yellow fluorescence of FVIIIYFP was no longer observed. These results demonstrate that FVa can block the FVIII endocytosis by CHO-LDLR+ cells. The observations together suggest that LDLR can not only bind FVIII but also FVa. In line with this notion, we established that CHO-ldlA cells, which lack functional expression of LDLR, were unable to internalize neither FVa nor FVIIIYFP. In addition, the ligand-binding clusters II and cluster IV of LRP effectively inhibited the endocytic uptake of FVa by CHO-LDLR+ cells. This implies that structural elements of FVa involved in LRP binding may overlap with those required for LDLR dependent internalization. Our results suggest a so far unidentified role for members of the LDL receptor family in the regulation of FVa in plasma.
AHSP Knockdown in Human Erythroleukemia Cell Line and Human Hematopoietic Stem Cells Results in Alpha Hemoglobin Chain Precipitation, Decreased Hemoglobin Levels and Increased Cell Death.
Authors: Pinho Flavia O. , Albuquerque Dulcineia M. , Saad Sara T.O , Costa Fernando F.
Published: April 2020

Alpha Hemoglobin Stabilizing Protein (AHSP) binds alpha hemoglobin chain (αHb), avoiding its precipitation and its pro-oxidant activity. In the presence of beta hemoglobin chain (βHb), the αHb-AHSP complex is dismembered and βHb displaces AHSP to generate the quaternary structure of hemoglobin. These data have been obtained in vitro and in mouse cells, but strongly suggest the importance of AHSP for normal hemoglobin synthesis in humans. To the best of our knowledge, the relationship between hemoglobin formation and alterations in AHSP expression has not yet been described in human red cells. Hence, to investigate the consequences of a reduced AHSP synthesis in human red cells, we established the RNA interference-mediated knockdown of AHSP expression in human erythroleukemia cell line (K562 cells) and human hematopoietic stem cells (CD34+ cells) induced to erythroid differentiation, and analyzed the consequent cellular and molecular aspects of AHSP knockdown in these cells. shRNA expression vectors, aimed at the AHSP mRNA target sequence, were cloned and transfected into K562 and CD34+ cells using a non-liposomal lipid reagent. Following transfection, K562 cells that stably expressed AHSP-shRNA and CD34+ cells that transiently expressed AHSP-shRNA were selected. K562 and CD34+ cells were stimulated to erythroid differentiation by hemin and erythropoietin (EPO) respectively. The cells were examined in terms of gene expression using quantitative real-time PCR; production of reactive oxygen species (ROS), apoptosis and hemoglobin production through flow cytometry assays; and immunofluorescence assays for globin chains. AHSP-shRNA hemin-induced K562 cells and AHSP-shRNA EPO-induced CD34+ cells presented 71% and 75% decreases in AHSP expression levels, respectively. The RNAi-mediated knockdown of AHSP expression resulted in a considerable αHb precipitation, as well as in a significant decrease in fetal hemoglobin formation. In addition, AHSP-knockdown cells demonstrated an increased ROS production and increased rate of apoptosis. These findings strengthen the hypothesis that AHSP stabilizes the alpha hemoglobin chain, avoiding its precipitation and its ability to generate ROS which implicate in cell death. Moreover, data indicate that AHSP may be highly significant for human hemoglobin formation and suggest that AHSP is a key chaperone protein during human erythropoiesis.
GVHD Abrogates T Cell Responses to Dendritic Cell Vaccines but Not Vaccine-Induced Proliferation.
Authors: Capitini Christian M. , Guimond Martin , Mackall Crystal L. , Fry Terry J.
Published: April 2020

BACKGROUND: Allogeneic bone marrow transplant (BMT) is a potent form of immunotherapy against hematopoietic and potentially solid tumors, but relapse of malignancy and Graft versus Host Disease (GVHD) remain a major cause of morbidity and mortality. Dendritic cell (DC) vaccines represent a potential strategy to enhance the potency and efficacy of the Graft versus Tumor (GVT) effect without exacerbating GVHD, but little is known regarding the impact of the allogeneic BMT environment on vaccine responses. Previous work in our lab has shown that DC can effectively expand tumor-antigen specific T cells following minor-mismatched allogeneic transplantation, resulting in delay of tumor growth. However, GVHD completely abrogates these responses. We hypothesized that the mechanism behind the loss of DC vaccine responses was through diminished T cell proliferation in the setting of GVHD.

METHODS: We established a minor histocompatibility antigen mismatched BMT model by transplanting B6 x C3H.SW (F1) thymectomized mice with either CD45.1+ B6 (allogeneic) or CD45.1+/45.2+ F1 (syngeneic) T cell-depleted bone marrow on day 0, followed by a donor lymphocyte infusion at day 14 to induce GVHD. On day 28, we adoptively transferred transgenic CD8+ (CD45.2, V beta 8.3+) or CD4+ (CD45.2, V beta 6+) T cells that only recognize components of the HY antigen to avoid cross-reactivity with allogeneic antigens. The T cells were labeled with CFSE prior to transfer to explore the impact of vaccine-induced proliferation in the presence or absence of GVHD.

RESULTS: In the allogeneic BMT setting, both CD8+ and CD4+ HY-transgenic T cells given with a DC vaccine begin proliferating 5 days (CD8+ 40%, CD4+ 65% divided) after adoptive transfer. The syngeneic BMT vaccinated control mice show similar kinetics (CD8+ 38%, CD4+ 65% divided), suggesting GVHD does not decrease T cell proliferation. Interestingly, although HY transgenic T cells do not normally undergo homeostatic peripheral expansion (HPE) during lymphopenia, the CD4+ T cells proliferate in lymph nodes even in the absence of a vaccine (50% divided), but this was not observed in the respective syngeneic BMT unvaccinated control mice (0% divided).

CONCLUSIONS: DC vaccines represent a strategy to improve the efficacy of the GVT response following allogeneic BMT, however, GVHD can abrogate DC vaccine responses. The negative impact of GVHD on vaccine responses does not appear to result from impaired DC vaccine-driven proliferation. Indeed, even in the absence of a vaccine, non-alloreactive CD4+ T cells proliferate via a mechanism that appears to be distinct from HPE. This data provides important insights towards optimizing antigen-specific vaccines administered in the allogeneic BMT setting.
Immunological and Clinical Responses in Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL) after RHAMM-R3 Peptide Vaccination.
Authors: Greiner Jochen , Schmitt Anita , Giannopoulos Krzysztof , Chen Jinfei , Goetz Marlies , Rojewski Markus , Ritter Gerd , Gnjatic Sascha , Guillaume Philippe , Ringhoffer Mark , Bommer Martin , Schlenk Richard F. , Liebisch Peter , Bunjes Donald , Shiku Hiroshi , Dohner Hartmut , Schmitt Michael
Published: April 2020

We initiated a phase I/II R3 peptide vaccination for patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) overexpressing the receptor for hyaluronic acid mediated motility (RHAMM). RHAMM is a leukemia associated antigen (LAA) that is strongly expressed in several hematological malignancies and induces humoral and cellular immune responses. In this study, patients with AML, MDS, MM or CLL were included with RHAMM expression but with a limited tumor load or a minimal residual disease. To date, 25 patients were enrolled. The first 12 patients were vaccinated with 300 mcg and further patients with 1000 mcg R3 peptide emulsified with the incomplete Freund’s adjuvant. The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Only mild drug-related adverse events were observed such as erythema and induration of the skin at the site of injection. Immunological analysis were performed using enzyme linked immunospot (ELISpot) assays for Interferon gamma and Granzyme B, tetramer staining and chromium release assays. We detected specific immune responses in 70% of patients. In most patients, we found an increase of CD8+/HLA-A2/RHAMM R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Moreover, we measured IL-2 and IL-10 levels in sera before and after vaccination. While IL-10 levels remained at a rather low level, we detected an increase of IL-2 in four of ten patients who showed also clinical responses. RHAMM transcripts in bone marrow and peripheral blood were quantified by real-time RT-PCR. Responding patients showed a decrease of RHAMM after vaccination. We detected positive clinical effects in several patients with myeloid disorders showing a reduction of blasts in the bone marrow. One MDS patient did not need any longer erythrocyte transfusions. Two patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies.