bluuebunny/crossref_metadata_2025 huggingface dataset:
EBV Associated-Pneumonia in Patients with Post-Transplant Lymphoproliferative Disease after Hematopoietic Stem Cell Transplantation
Authors: Liu Qifa
Published: April 2020

Background. Traditionally, reactivation or infection of Epstein-Barr virus (EBV) after hematopoietic stem cell transplantation (HSCT) most often induces post-transplant lymphoproliferative disease (PTLD). In recent years, it is reported that it may manifest in patterns other than PTLD.

Methods. To investigate PTLD and PTLD accompanied by EBV associated- pneumonia among 323 patients after HSCT and discribe the clinical manifestation of PTLD accompanied by EBV associated-pneumonia.

Results. Seven cases of PTLD with primary presentation as extravisceral lymphoid tissue were identified. Five cases were diagnosed of CD20+ diffuse large B-cell lymphoma, 1 cases CD20+ polymorphic B-cell hyperplasia and 1 case CD3+CD45RO+ peripherial T-cell lymphoma-unspecified. Six patients were associated with EBV and 1 case was not in 7 cases of PTLD. Three patients were diagnosed of EBV associated-pneumonia and chest computed tomography revealed multifocal patchy and diffuse ground-glass attenuation in both lungs. EBV-DNA was positive in bronchoalveolar lavage fluid. There were mainly CD3+ T cells with the absence of CD19+ and CD20+ B cells in cytology analysis of BAL fluid. Lung biopsy showed that patients had interstitial and intra-alveolus infiltrates of main CD3+ T cells and part of CD68+ macrophages without CD19+ and CD20+ B cells. The patients with PTLD accompanied by EBV associated-pneumonia had hyperpyrexia and dyspnea. The diseases aggravated rapidly and eventually all died within two weeks since the onset of PTLD.

Conclusion. It is not rare for EBV associated-PTLD accompanied by EBV associated- pneumonia after HSCT. Cytology of BAL fluid and lung biopsy may help to the diagnosis.
QuantiFERON®-CMV for Immune-Monitoring in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Morello Enrico , Pagani Elisabetta , Monsurrò Vladia , Cavattoni Irene , Coin Silvia , Larcher Clara , Tridente Giuseppe , Cortelazzo Sergio
Published: April 2020

Background: CMV infection represents a major complication of allo-SCT affecting transplant related mortality and morbidity. Anti-viral therapy is toxic and prolonged treatment could affect graft function. Monitoring specific immune response against CMV could optimize the timing for anti-viral therapy administration in order to avoid related toxicity and to reduce CMV related mortality and morbidity. A recent ELISA based test (QuantiFERON®-CMV) could measure specific (anti-HCMV) and aspecific production of IFN-γ in whole blood.

Aims: to test the reliability of QuantiFERON®-CMV in a cross sectional study in order to identify patients at risk of CMV disease after alloHSCT.

Methods: QuantiFERON®-CMV is an in vitro diagnostic test using a peptide cocktail simulating human cytomegalovirus proteins (CMV) to stimulate cells in heparinised whole blood. Detection of interferon-γ (IFN-γ) by ELISA is used to identify in vitro responses to these peptide antigens that are associated with CMV infection. The IFN-γ response in the CMV Ag tube is considered positive if > 0.2 UI/mL as defined by the manufacturer. The mitogen-stimulated plasma sample was used as an IFN-γ positive control (PC) for each specimen tested. CMV reactivation and disease were defined according EBMT recommendations.

Results: Among 92 tests no correlation between pp65 antigenemia and IFN-γ production was proved (p=0,346). However, among the 41 tests showing lower levels of anti-HCMV IFN-γ production (<0.2 IU/mL) 8 tests belonging 4 patients were associated with CMV disease, whereas among the 51 tests showing higher levels of anti-HCMV IFN-γ production (>=0.2 IU/mL) none were associated with CMV disease (p=0.001), RR 2.5 (CI95% 1.951–3.321).

Conclusions: QuantiFERON®-CMV doesn’t seem to represent a significant reliable test for risk of viremia after alloHSCT, but patients with prolonged lower levels of anti-HCMV IFN-γ production (<0.2 IU/mL) are at risk of CMV disease. Prospective studies are required in order to identify the correlation between viremia and the need for treatment.
Reduced Intensity Conditioning (RIC) Allogeneic Stem Cell Transplantation (allo-SCT) in Patients with Multiple Myeloma (MM): Long Term Follow-up in a Single Centre Series
Authors: Benakli Malek , Ahmednacer Redhouane , Talbi Amina , Mehdid Farih , Belhadj Rachida , Rahmoune Nadia , Harieche Farida , Zerhouni Fatiha , Hamladji Rose-Marie
Published: April 2020

Background: RIC allo-SCT has been proposed as a strategy for retaining the graft versus myeloma effect of allo-SCT while decreasing transplant related mortality (TRM). Here, we retrospectively studied a series of 15 patients (pts) with MM treated by RIC allo-SCT.

Patients and methods: Between April 2001 and December 2007, 15 pts with MM underwent RIC allo-SCT with an HLA-identical sibling donor. Initially, 8 pts had MM with Ig G, one IgA, 2 light chains, 3 non-secretory and one undetermined. Three pts were stage II and 12 stages III. At time of allo-SCT, 6 pts were in complete remission and 9 in refractory/progressive disease (2 received prior autologous transplants). Median age was 48 years (range, 38–60) and the sex-ratio (M/F) 1,5. Median time from diagnosis to RIC allo-SCT was 18 (range, 6–76) months. The conditioning regimen included Fludarabine 150mg/m2 and Melphalan 140mg/m2. GVHD prophylaxis consisted of association cyclosporine (cSA) and mycophenolate (MMF). All pts received G-CSF mobilised peripheral blood stem cells, with a median CD34+ cell count: 4,5.106/kg (range, 1.92–13).

Results: Neutropenia occurred in all pts and the median duration of aplasia was 8 (range, 5–14) days. Only 3 pts (20 %) required red blood cells transfusions and 12 pts (80 %) needed platelets transfusions. Acute GVHD was observed in 6 cases (40 %) including 4 cases of grade II–IV. Eight pts (72 %) had chronic GVHD, of whom 5 with an extensive form. Three pts (20 %) had CMV reactivation at a median time 91 (range, 53–158) days after transplantation. Four pts (26 %) had late onset relapse at a median time of 826 (range, 248–1370) days. TRM was 33 % at one year after RIC allo-SCT. With a median follow-up of 50 (range 14–86) months, 5 pts (33 %) are still alive in complete remission with full donor chimerism. Ten pts (66 %) died (2 early severe infections, 3 acute GVHD, 3 after relapse, one myocardial infarction, and one public highway accident). Overall and progression-free survivals at 7 years are 37,5 % and 31,2 % respectively.

Conclusion: This study suggests that RIC allo-SCT is a potential therapy for relapsed MM. However, TRM and relapse remain a matter of concern, likely due to the inclusion of refractory and heavily pretreated pts with many comorbid conditions. Future protocols, should aim for better patient selection, focussing on those pts in first chemosensitive relapse.
Hematopoietic Stem Cell Defects in Novel Fanconi Anemia Mouse Models
Authors: Parmar Kalindi , Kim Jung Min , Shimamura Akiko , Stuckert Anna , DeLoach Mary K , Zhu Kaya , Mauch Peter M. , Kutok Jeffery , Aster Jon , Akashi Koichi , Gilliland D. Gary , D’Andrea Alan D.
Published: April 2020

Fanconi Anemia (FA) is an autosomal recessive disease characterized by congenital defects, crosslinker hypersensitivity, progressive bone marrow failure, and a defect in hematopoietic stem cell (HSC) function. There are thirteen known Fanconi Anemia genes (A, B, C, D1, D2, E, F, G, I, J, L, M, N) and disruption of these genes appears to account for a variable degree of HSC dysfunction. Another gene (Usp1), encoding a deubiquitinating enzyme which is not a FA gene per se, is a critical regulator of the FA pathway. Understanding the mechanism of action of the FA proteins may allow better diagnosis and treatment for Fanconi Anemia and other aplastic anemia patients. Here, we have characterized mouse models for FA, including Fancd2−/− and Usp1 −/− mice. Similar to a previously reported Fancd2 mouse model (Houghtaling S et al, Genes & Development, 17:2021–2035, 2003), the Fancd2−/− mice developed in our laboratory displayed FA phenotypes including cellular hypersensitivity to mitomycin C (MMC), hypogonadism and reduced fertility. Interestingly, Usp1−/− mice developed for the first time in our laboratory exhibited severe FA phenotypes, with increased cellular hypersensitivity to MMC, 80% perinatal lethality, testicular atrophy, and depletion of germ cells. Quantitative assessment of the Fancd2 protein expression in the bone marrow using the AQUA imaging system demonstrated low levels of Fancd2 staining in the Lin+ cells compared to the Lin− cells in wild-type (WT) mice, with an absence of Fancd2 expression in the Fancd2 −/− mice. Analysis of hematopoietic parameters in both the Fancd2 and Usp1 mouse models showed normal white blood cell counts and normal hemoglobin levels in the peripheral blood. However, bone marrow from Fancd2−/− mice as well as Usp1−/− mice exhibited phenotypic and functional differences compared to the bone marrow from WT mice. Multiparameter flow cytometric analysis of bone marrow revealed increased frequencies of MEPs (megakaryocyte-erythroid progenitors) in Usp1−/− mice and decreased frequencies of LSK (Lin-Sca-1+Kit+) population in Fancd2−/− mice in comparison to WT controls. In addition, bone marrow from both Fancd2−/− mice and Usp1−/− mice contained significantly reduced frequencies of late-developing day 28 CAFCs (cobblestone area-forming cells) compared to the bone marrow from WT mice. Furthermore, lethally irradiated recipients transplanted with bone marrow from Fancd2−/− or Usp1−/− mice showed reduced donor-type blood cell chimerism at 16 weeks post-transplant, compared to the recipients transplanted with WT bone marrow. Collectively, our data indicate that mice deficient in Fancd2 or Usp1 have hematopoietic stem cell defects and that these mouse models can be useful for development of therapeutics for FA.
Unrelated Donor Hematopoietic Stem Cell Transplantation for Treatment of High-Risk Leukemia
Authors: Depei Wu , Tang Xiao wen , Sun Ai ning , Fu Zheng -zheng , Miao Miao , Jin zheng Ming , Qiu Hui ying , Chang Wei rong , Zhu Xia ming
Published: April 2020

objective: To investigate the efficacy of unrelated donor hematopoietic stem cell transplantation(URD-HSCT) for high-risk leukemia.

Methods: From June 2001 to July 2008, 62 patients of high-risk leukemia underwent HSCT with unrelated donor. The median age was 24(6~49)years. There were 21 unrelated donor’s grafts came from Buddhist Tzu Chi Stem Cells center in Taiwan, and 41 unrelated grafts came from Red Cross Society of China Hematopoietic Stem Cell Marrow Donor Program Administration Center. Seventeen patients received bone marrow transplantation and another 45 patients received peripheral blood stem cell transplantation. Fifty patients were fully matched with their donors for HLA loci A and B as well as for HLA-DRB1. Eight of 62 patients had 1 molecular loci mismatch and 4 of 62 patients had 2 molecular locus mismatch. The conditioning regimen consisted of modified BU/CY or modified total body irradiation(TBI) plus CY. Eleven patients received cyclosporine (CSA), short-term methotrexate (MTX) and mycophenolate mofetil (MMF) as the regimen of prophylaxis of GVHD. Forty |four patients received CSA, MTX and MMF plus ATG/ALG for GVHD prophylaxis. The combination of CSA, MTX, MMF,ATG/ALG and CD25 monoclonal antibody for preventing GVHD in 4 patients. Other two patients received Tacrolimus, short-term MTX and ATG to prevent GVHD.

Results: The incidence and severity of regimen-related toxicity were mild. The median number of MNC and CD34 positive cell was 4.48×108/kg and 4.06×106/kg respectively. The median time of the engraftment of neutrophil and platelet was 13 days and 16 days posttransplant respectively. The incidence of infection posttransplant was 48%. The complicated pneumonitis occurred frequently (67%), The incidence of grade I–II and grade III–IV acute GVHD was 17% and 23% respectively. Limited chronic GVHD occurred in 17% patients and extensive cGVHD presented in 14% patients. Median follow up was 6 (range 1 `85) months after transplantation. 83% patients achieved full donor chimerism(FDC) detected by STR-PCR and FISH. While 17% patients presented mixed chimerism (MC) and 10 of them converted to unstable MC. Among them 4 patients presented graft rejection and 6 patients suffered from relapse. Decreasing values of donor chimerism were detected prior to the relapse of disease. Furthermore the treatment related mortality (TRM) was 16%(10/62), The incidence of relapse was 10%. Until now 37 patients are still alive. The 5-years expected survival was 52.2%.

Conclusion: URD-HSCT can be an effective and curable approach for high risk leukemia with higher incidence of GVHD and infection. The treatment for the severe and deadly GVHD and pulmonary infection are still major problems need to be resolved in the future.
Unrelated Cord Blood Transplants in Children with Acute Leukemia: Experience of a Single Center
Authors: Ferreira Rosa Branca , Vaz Carlos Pinho , Barbosa Isabel , Roncon Susana , Campilho Fernando , Carvalhais Alzira , Martinho A. , Campos Antonio
Published: April 2020

Allogeneic stem cell transplantation is an accepted treatment modality for selected malignant diseases. However, the ability to identify suitable related or unrelated donors can be difficult in some patients (pts). Alternative sources of stem cells such as cord blood (UCB) provide a readily available graft mainly for pediatric patients. Between October 1996 and May 2008, 23 consecutive patients (pts), with a median age of 6 years old (range:1 – 11), 13 male and 10 female, underwent UCB transplant for the treatment of acute lymphoblastic leukemia (n= 15), acute myeloid leukemia (n=7) and acute byphenotypic leukemia (n= 1). Conditioning regimen consisted of intravenous busulfan (3.2 mg/kg/day x 4 days), cyclophosphamide (120 mg/Kg) and rabbit antithymocyte globuline (15 mg/Kg) ±melphalan (140 mg/m2) and GVHD prophylaxis consisted of a calcineurin inhibitor and MMF or MTX. All grafts were HLA mismatched. The median infused cell doses were, respectively 3.2× 107/kg (range: 1.2–21) and 1.27×105/kg (range:0.2–4.3) for nucleated cells (NC) and CD34 + cells. Engraftment occurred in 18 pts (75%). Five pts failed engraftment, of wich 4 underwent a second transplant (3 autologous and 1 haploidentical) and 1 died of infection at day +59. Grade II–IV acute GVHD occurred in 12 pt and chronic GVHD in 2 of 14 pt at risk. With a median follow up of 1,5 years (range: 3 months-9.5 years), overall survival (OS) at 3 years is 40% (± 13%) and event free survival (EFS) at 3 years is 32% (± 11%). Non relapse mortality (NRM) was 13.6 % (± 7.3%). UCB transplants represents a valuable alternative to bone marrow or peripheral blood especially for pts requiring urgent transplant or lacking an HLA matched unrelated donor and having the potential advantage of an immune tolerance allowing successful transplantation despite HLA disparity
Allogeneic Stem Cell Transplantation Following Reduced Intensity Conditioning Regimen for Treatment of Refractory Leukemia
Authors: Jiang Jieling , Wang Chun , Yan Shike
Published: April 2020

Objective To evaluate the efficacy of allogeneic stem cell transplantation (allo-HSCT) following reduced intensity conditioning (RIC) regimen in the treatment of relapsed and refractory leukemia.

Methods Fourteen patients with acute myeloid leukemia, eleven patients with acute lymphocyte leukemia and two patients with chronic myeloid leukemia blast phase received allo-HSCT following RIC regimen consisting of fludarabine and small or moderate dose total body irradiation (TBI). Patients received mobilized peripheral stem cell from HLA matched siblings (n=11), at least 5/6 matched unrelated donor (n=10) or haploidentical related donor (n=6). All patients were in advanced disease before transplantation. Graft versus host disease (GVHD) prophylaxis program consist of cyclosporin A plus mycophenolate mofetil or short-term methotrexate, or these three drugs combination; CD25 monoclone antibody and ATG were also used in patients with unrelated or haploidentical donors.

Results Sustained engraftment was attained in 22 patients, the median time to ANC >0.5×109/L was 13 days (range: 11~17days), and the median time to BPC > 50×109/L was 19 days (range: 12~42days). Detected by short tandem repeat (STR)-PCR, complete donor chimerism was comfirmed in 20 patients with a median of 14days (range: 7~70 days). With a median follow-up of 9 months (range, 1~44months), the incidences of acute GVHD and chronic GVHD were 50% (11/22) and 41.2% (8/17) respectively. The transplant related mortality was 25.9% (7/27), mainly from graft failure (n=4), intracranial hemorrhage (n=1), acute GVHD (n=1), and severe infection(n=1). At the time of last follow up, ten patients relapsed, eleven patients were alive and leukemia free. Probabilities of overall survival for AML and ALL patients were (51.9±13.4)% and (32.7±15.0)% at 2 years, respectively. It seems that AML patients had a better outcome than ALL patients, but there was no significant difference (289).

Conclusion Allogeneic stem cell transplantation following fludarabine and TBI based RIC regimen could be used in the treatment of relapsed and refractory leukemia with well tolerance and low transplant related mortality from which patients may be benefit.
High Probability of Survival after Related and Alternate Donor Hematopoietic Cell Transplantation for Pediatric Patients with High Risk AML Using a Fludarabine Based Preparative Regimen
Authors: Tan Poh-Lin , Yeoh Allen , Quah Thuan-Chong , Kurtzberg Joanne , Wagner John E.
Published: April 2020

Hematopoietic cell transplantation (HCT) in pediatric patients with high risk acute myeloid leukemia (AML) such as those with non-Down de novo acute megakaryoblastic leukemia (AMKL), transformed myelodysplastic syndrome (MDS), therapy-related AML (tAML) and refractory/resistant AML (rAML) has been limited by graft failure, regimen related toxicity (RRT) and relapse. Between September 2005 and July 2008, 10 pediatric patients, median age of 7.6 (range, 1.3 – 17.0) years with such high risk AML (AMKL, n=2; MDS/AML, n=4; tAML, n=1; rAML; n=3) received a novel Fludarabinebased preparative regimen followed by T cell replete transplantation with bone marrow (BM, n=4), peripheral blood stem cell (PBSC, n=5) or umbilical cord blood (UCB, n=1) in a Singapore Pediatric BMT center. Seven patients received stem cells from alternate donors (AD) and 3 from matched related donors (MRD). Prior to HCT, 6 patients were in 1st complete remission (CR1), 2 in CR2 and 2 had progressive diseases. AD consisted of single antigen mismatched related and non-genoidentical unrelated donors (antigenic match at HLA-A, B, DRB1*). All patients received IV FLU 125 mg/m2, BU, 0.8 – 1.2 mg/kg 6 hourly, MEL, 140mg/m2. AD recipients received additional ATG (Thymoglobuline) 5mg/kg. GVHD prophylaxis consisted of cyclosporine and methotrexate or mycophenolate motfetil depending on the stem cell source. Excluding the 2 patients who entered HCT with progressive diseases, all other patients achieved myeloid engraftment at a median of 14 (range, 9 – 16) days and achieved sustained stable full donor chimera from 21 days after HCT. The median duration and costs of HCT admissions was 34 (range, 17 – 66) days and USD 51,317 (range, 41,416 – 155,905), respectively. Common post-HCT complications included engraftment syndrome (n=4) and reactivation of Herpes viruses (n=4). There was no severe RRT. Incidences of grade I–III acute GVHD and chronic GVHD were 63% and 67%, respectively. All these patients are alive in sustained CR at a median follow-up of 303 (range, 44 – 1037) days with Karnosky/Lansky scores of 90 – 100%. Both the patients with progressive diseases had graft failures and subsequently died of disease at a median of 138 (range, 77 – 198) days after HCT despite experimental salvage therapy with allogeneic natural killer cell transplantation from parental donors. In summary, this novel FLU based preparative regimen paired with non-genoidentical HCT is safe, inexpensive, allows prompt engraftment, and appears to provide anti-leukemia effects in pediatric patients with high risk AML entering HCT in remission. Long term follow-up will determine relapse risks and whether this non-irradiation based regimen is associated with a reduction in late effects including malignancy.
Laser Transmyocardial Revascularization (TMR) Combined with Autologous Bone Marrow Cells for Diffuse Coronary Disease: Immunophenotype Analysis and Preliminary Clinical Results in 14 Cases
Authors: Alegre A. , Aguado B. , Alfonso-Pérez M. , Muñoz C. , de Landázuri M. Ortiz , Sánchez-Madrid F. , Cámara C. , Lara F. , Duarte J. , Reyes G.
Published: April 2020

Introduction: Stem cell (SC) based therapy in ischemic heart disease is actually in development with the aim of improving cardiac regeneration. The way of administration, source of SC and the mechanisms of its clinical effects are yet not well defined. We present preliminary results of a group of patients treated with laser revascularization and autologous bone marrow (BM) cells administration, evaluating cells immunophenotype and clinical results.

Patients and methods: Fourteen patients (57–78 y) with diffuse coronary disease and medically refractory class III/IV angina were prospectively evaluated for cell-based therapy combined with laser transmyocardial revascularization (TMR) PHOENIX-TM (Cardiogenesis, Irvine, CA, USA). BM mononuclear cells (MC), were obtained by BM aspiration (120 mL) and processed for concentration using HARVEST-TM system (Harvest Technologies Inc. GMBH, Munich). Immunophenotypic characterization of the final infused product was performed by standard four-color flow cytometry with monoclonal antibodies directed against the human surface antigens CD29, CD49a, CD105, CD106, CD117, CD34, CD133 and CD45, to evaluate different cell populations.

Results and conclusions: Patients received a median of 56.8×106 (43.7–79) BMMC/ mL 0.5×106 (0.05–1) CD34+ cells/mL and 0.23×106 (0.001–0.7) CD133+ cells/mL. All the patients are alive after this treatment: median of 9 months, (1–15), with improvement of their cardiological clinical status and QoL. As observed (Table 1) the BMMC used in the therapy contained a significant proportion of CD133+ and CD34+ hematopoietic SC. In addition, the samples contained cells with CD105+ CD45− or CD29+ CD45− CD34− phenotypes which have been described as mesenchymal stem cells (MSC). Bone marrow CD106+ positive cells represent a multipotent subpopulation of MSC including endotelial. Correlation between the relative quantity of the different cell subsets and clinical results of the patients will be presented.

TABLE 1

CD34 CD133 CD117 CD29 CD49a CD105 CD106 Mean 3.53 2.42 7.37 2.23 0.75 6.75 0.33 Std. Deviation 1.72 1.68 2.60 0.97 0.75 3.34 0.23 25% Percentile 1.95 0.85 4.95 1.25 0.15 3.10 0.2 Median 3.20 2.15 6.70 2.30 0.40 7.05 0.25 75% Percentile 5.45 4.25 10.45 3.15 1.70 10.10 0.55
Mini-Autologous Hematopoietic Stem Cell Transplantations as a New Treatment Modality for Multiple Sclerosis Patients
Authors: Novik Andrei A , Melnichenko Vladimir Y , Fedorenko Denis A , Kruglina Ruslana V , Ionova Tatiana I , Kishtovich Anton V , Ivanov Roman I , Gorodokin Gary J
Published: April 2020

Conventional therapies do not provide satisfactory control of multiple sclerosis (MS) due to their inability to eradicate self-specific T cell clones. Recently, high-dose immunosuppressive therapy with autologous hematopoietic stem cell transplantation (AHSCT) was proposed as a new and promising therapy for MS patients. Taking into account the information about high risk of transplant-related mortality and severe side effects of myeloablative conditioning regimens, the rationale to use non-myeloablative regimens sounds reasonable. The goal of our research was to study the safety and efficacy of mini-AHSCT in MS patients. Fifty three patients with MS (secondary progressive – 16 patients, primary progressive – 12, progressive-relapsing – 3 and relapsing-remitting – 22) were included in this study (mean age - 30.0, range: 17–47; male/female – 23/30). The conditioning regimen included reduced or modified BEAM. Median EDSS at base-line was 4.0 (range 1.5 – 8.0). The median follow-up duration was 7 months (range 6 – 20 months). Neurological and quality of Life (QoL) evaluation was performed at baseline, at discharge, at 3, 6, 9, 12 months, and every 6 months thereafter AHSCT. MRI examinations were performed at baseline, at 6, 12 months, and at the end of follow-up. Transplantation procedure was well tolerated by the patients with no transplant-related deaths. Among 29 patients included in the efficacy analysis 15 patients (52%) experienced clinical stabilization; 14 (48%) – improvement. EDSS improved by 0.5 points in 11 patients, by 1.0 points in 2 patients, by 2.0 points in 1 patient. One patient progressed after 6 months stabilization. No active, new or enlarging lesions were registered in patients without disease progression. Out of 27 patients included in QoL analysis 21 exhibited QoL improvement and 4 – stabilization during the follow-up; QoL worsened in 2 patients. All the patients without disease progression were off therapy throughout the post-transplant period. In conclusion, mini-AHSCT may be considered as a new, safe and effective treatment for MS. The results of mini-AHSCT in MS patients are promising both in terms of clinical and patient-reported outcomes. The collection of long-term follow-up data is worthwhile to verify these findings. The rationale of evolution from myeloablative to non-myeloablative transplant regimens should be confirmed by the further studies.
Genome-Wide Scanning of Epigenomic Alterations in Childhood Leukemia
Authors: Zhong Sheng L. , Xiao Yuanyuan , Yeh Ru-Fang , Zhou Mi , Wrensch Margaret R , Zheng Shichun , Wiencke John , Wiemels Joseph L.
Published: April 2020

To study how epigenetic changes contribute to childhood leukemia, and to identify new disease biomarkers, we combined an approach of pharmacologic inhibition of epigenetic modifications of chromatin with gene expression arrays (Affymetrix Exon 1.0; and miRNA array, UCSF-designed) to reveal precise epigenetic aberrations on a global scale in defined subsets of leukemia cell lines that recapitulate common genetic subtypes of leukemia. From these analyses, we identified >200 genes up-regulated by 5-aza-2′-deoxycytidine and trichostatin A treatment (including genes encoding both mRNA and miRNA). We validated 50 selected genes using real-time quantitative PCR. We also tested promoter regions for methylation in the cell lines and selected pediatric leukemia bone marrows using bisulfite-DNA sequencing. A panel of leukemia-specific methylation targets was discovered, which includes some well-known genes (such as HOXA5 and DAPK) and several novel candidates in childhood leukemia (eg., CD82 and KLF4). To extend these observations, we performed methylation microarrays on DNA samples isolated from the studied cell lines as well as bone marrows of 35 childhood leukemia patients with defined cytogenetics, using Illumina Methylation Cancer Panel I. This panel includes a total of 1,505 CpG loci from 807 genes. We also assayed corresponding archived newborn bloods (Guthrie cards) from 32 cases, and 19 control Guthrie cards of children matched to the cases based on gender and age, to guage the impact of congenital methylation events on childhood leukemia. Many targets genes, including known tumor suppressor genes and genes involved in DNA repair, cell cycle control and differentiation were found to be methylated in leukemia cell lines and the leukemic bone marrow samples, but not in Guthrie card controls. In addition, the methylation status of a core group of 67 CpG sites can differentiate the common subtypes of childhood leukemia – those with TEL-AML1, E2A-PBX1, high hyperdiploidy, and normal karyotypes. Several genes concordantly methylated in Guthrie cards and leukemic bone marrow may represent prenatal epimutations. With the integration of such assays, we have constructed a panel of potential candidate genes and miRNAs involved in development of childhood leukemia which have aberrant promoter methylation. Our studies lay a foundation for a comprehensive understanding of the full extent of epigenetic changes in development of childhood leukemia and how they may be exploited for therapeutic purposes as well as for early detection of this disease.
Differente Patterns of Telomerase Reverse Transcriptase (hTERT) mRNA Variant Expression Profiles in Patients with Myelodysplastic Syndromes (MDS) and Acute Myelogenous Leukemia (AML)
Authors: Verrou Evgenia , Kaloutsi Vassiliki , Katodritou Eirini , Garypidou Vasilia , Nikitidou Anna , Gastari Vassiliki , Lazaridou Anna , Zervas Konstantinos , Kotoula Vassiliki
Published: April 2020

BACKGROUND: Alternative splicing of hTERT mRNA contributes to the regulation of telomerase activity in normal and neoplastic cells. The deleted variant A (Adel), lacking 36nts in the beginning of exon 6, blocks telomerase activity while the B deleted variant (Bdel), lacking exons 7 and 8, exhibits no action in terms of telomerase activation. The expression of hTERT mRNA has been investigated in myeloid malignancies with controversial results. The expression of hTERT mRNA variants has not been explored in MDS patients.

AIM: To investigate the expression of hTERT mRNA variant transcripts A+B+ (contained in the full-length product), Adel and Bdel in the bone marrow of MDS and AML untreated patients.

METHODS: Bone marrow aspirates from 27 patients with refractory anaemia or refractory cytopenia with multilineage dysplasia (RA or RCMD, defined as group A), 13 patients with refractory anaemia with excess of blasts (RAEB1 or RAEB2, defined as group B) and 17 AML patients (defined as group C) were studied. Relative expression of the above transcripts was assessed on total RNA with reverse transcription followed by real-time polymerase chain reaction (PCR). The data obtained were analyzed by using Pearson chi-square and Kruskal Wallis statistics. All individuals signed informed consent before sampling.

RESULTS: hTERT mRNA was detected in 10, 4 and 3 patients of groups A, B and C respectively. The full length product (A+B+) was detected in groups A, B and C (2/27, 4/13, 3/17 respectively) (pA/B=0.053). Adel isoform was detected in groups A (7/27) and B (3/13) but not in group C (0/17) (pA/C=0.022). Bdel isoform was observed in group A (3/27), B (3/13) and C (2/17). Co-expression of isoforms was observed in 2 patients of A group (Adel/Bdel, Adel/A+B+), 4 patients of group B (1 Adel/A+B+, 1 Bdel/A+B+, 2 Adel/Bdel/A+B+) and two patients of group C (Bdel/A+B+). Relative expression levels of Adel, Bdel, and A+B+ transcripts were not significantly different among patient groups.

CONCLUSIONS: Alternatively spliced hTERT variants either lack a critical reverse-transcriptase motif or produce a non-functional reverse-transcriptase. Therefore, cells may control telomerase activity by switching their hTERT mRNA variant expression profile. Our results indicate that aberrations in hTERT variant profiles, especially regarding the Adel isoform, may be implicated in the pathogenesis and progression of MDS. The increased incidence of Adel positivity among RA or RCMD patients may be connected with a decline in telomerase activity and therefore contribute to the establishment of chromosomal abnormalities. This observation underlines the need to distinguish between the different hTERT mRNA variants when investigating hTERT expression in MDS patients.
Upregulation of hGC-1 Expression by Hypomethylation, Retinoic Acid and Interferons in Human Myeloid Leukemia
Authors: Liu Wenli , Rodgers Griffin P
Published: April 2020

hGC-1 (Granulocyte colony stimulating factor induced gene-1, also called GW112 and olfactomedin 4) was first identified in human myeloid precursor cells induced by granulocyte colony stimulating factor (G-CSF). It is a myeloid lineage and differentiation stage specific gene. Its expression, regulation and biological function, specifically in myeloid cells, are still poorly understood. In this study, we analyzed the hGC-1 gene expression in leukemia patients and further investigated the mechanism of hGC-1 gene regulation in leukemia cells. We found that hGC-1 was overexpressed in myeloid leukemia patients compared with normal individuals in peripheral blood leukocytes (p<0.01) and its expression in accelerated phase of chronic myeloid leukemia (CML) patients was significantly higher than that in chronic phase (p<0.01) using a dot blot and quantitative RT-PCR analysis. Hypomethylation of CpG sites in the promoter of hGC-1 gene were observed in CML patients by pyrosequence and 5-aza-2′-deoxycytidine induced hGC-1 expression in myeloid leukemia cells, suggesting that promoter CpG methylation status affects the expression of hGC-1 gene. All-trans-retinoic acid (ATRA) and interferons (IFNs) are active anti-leukemia agents. ATRA and IFNs have shown synergistic interactions in various experimental conditions and represent a potentially useful therapeutic combination in the treatment of various types of leukemia. However, the target genes and molecular basis of these interactions still needs to be further elucidated. Here, we identified that hGC-1 was a target gene of RA in myeloblastic leukemia cells. Treatment with ATRA induced hGC-1 expression in HL-60 cells and enhanced hGC-1 expression in AML-193 and GDM-1 cells. Deletion analysis led to the identification of a positive retinoic acid response element (DR5) and a negative response element (DR1) within hGC-1 promoter. Furthermore, electrophoretic mobility-shift assays demonstrated that RARa/RXRa binds to the DR5 site. Transfection study in COS-7 cells revealed RARa/RXRa mediated the RA induced transactivation of hGC-1 promoter. We also found that hGC-1 was an early responsive gene of IFN a and b in myeloid leukemia cells (HL-60, AML-193 and GDM-1). An effective interferon-stimulated response element (ISRE) was identified in the promoter of hGC-1 gene by examining the deletion mutants in luciferase reporter gene assay. Combined application of ATRA and IFNa and IFNb enhanced hGC-1 expression synergistically. Taken together, hGC-1 is identified as a novel target gene of methylation modification, RA and IFNs in myeloid leukemia cells. Our results suggest that hGC-1 is a potential marker for CML stage and may play a role in retinoic acid and interferon induced biological effects in leukemia cells.
Clinical Significance of P16 in Langerhans Cell Histiocytosis
Authors: Kim Sun Young , Kim Hee Jin , Yoon Hoi Soo , Bae Keun Wook , Park Jeong A. , Im Ho Joon , Seo Jong Jin , Lee Mee Jeong
Published: April 2020

Background. The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number of human cancers, emphasizing its importance in regulating cell cycle progression. The authors have chosen to analyze P16 expression in biopsies from children with LCH and correlated it with clinical manifestation and outcome in a broad range of organs.

Patients and Methods: Archival paraffin block were retrieved from children diagnosed with LCH and followed up at Asan medical center and Chungnam National University Hospital between 1998 and 2007. Pathological Langerhans cells were identified morphologically associated with the characteristic inflammatory type infiltrate and were confirmed by immunohistochemical staining with antibodies to CD1a, and S100. The disease was classified as single system defined as uni- or multifocal involvement of a single organ system or multi-system defined as involvement of multiple organ systems with or without organ dysfunction. The slides were stained with P16 antibody and semi-quantitatively evaluated using 5 grade systems, negative means absence of staining, ±, weakly positive, +, similar with lymphocytes around the LCs, 2+, stronger than lymphocystes, 3+, much stronger than lymphocytes.

Results: The specimens from 57 patients (31 boys and 26 girls) were available for immunohistochemistry. Of these, 12 slides were not suitable because the quality of staining was not good for evaluation or the tumor cells are too difficult to be differentiated. The median age of 45 patients (24 boys and 21 girls) was 9.5 years, range 5 months to 22 years with a definite diagnosis of LCH based on CD1a positivity. The P16 protein was expressed in various degrees except one specimen. No significant difference was found between the level of P16 expression in lesions from patients with single-system, and multi-system disease. But higher tendency of relapse have shown in 1+, 2+ and 3+ groups compared with negative and weakly positive groups.

Conclusions: This study has shown a diffuse expression of P16 protein in both single and multi-system lesions of LCH. P16 might be involved in the pathogenesis of LCH.
GATA-1 Protein Levels Are Not Altered in CD34+ and CD61+ Cells, but NF-κb Protein Levels Are Elevated in Patients with Idiopathic Myelofibrosis
Authors: Wang Jen-Chin , Naik Seema , Chen Chi , Xiao Ying-Yi , Chang Tsong H.
Published: April 2020

GATA-1 and NF-kB are important in megakaryocytopoiesis and pathogenesis of idiopathic myelofibrosis (IMF). Previous studies have shown that GATA-1 levels were normal in mRNA but were lower in protein content in CD61+ cells of patients with IMF, and we reported previously that GATA-1 protein levels were not altered in CD61+ cells. The current study includes more patients and also measured NF-kB levels. ELISA assay (GATA-1 and NF-kB ELISA kit, Panomics Inc. CA) was used to determine and compare the protein content of nuclear extracts of blood hematopoietic progenitor cells (CD34+) and megakaryocytes (CD61+ cells) from patients with IMF, patients with other myeloproliferative disorders, and with controls. CD61+ cells were obtained from 10-day cultures of blood CD34+ cells in serum-free culture medium with thrombopoietin and stem cell growth factor. Our study confirmed that GATA-1 protein levels are not altered in megakaryocytes (cultured CD 61+ cells) in patients with IMF (Fig. 1) and NF-kB levels are increased (Table 1). We conclude that GATA-1 protein levels are not altered in patients with IMF compared with controls.

Fig 1. GATA-1 Protein levels in CD61+ cells Cultured from Patients with IMF & Others Fig 1. GATA-1 Protein levels in CD61+ cells Cultured from Patients with IMF & Others

Table 1. NF-kB Levels (ng/2μg protein)

IMF Controls CD34+ cells 1.39 ± 0.27 1.10 ± 0.24 CD61+ cells 1.54 ± 0.52 0.45 ± 0.22
Longterm Prophylactic Treatment with Recombinant Factor VIIa (NovoSeven) in Severe Congenital Factor VII Deficient Patients
Authors: Gerhardt Andrea , Scharf Rudiger E. , Zotz Rainer B.
Published: April 2020

Background: Treatment of congenital FVII deficiency consists of replacement therapy with plasmic FVII concentrates or recombinant FVIIa (rFVIIa). Relatively small amounts of rFVIIa are required for replacement therapy in FVII deficient patients. Because of its short half-life of approximately 3 hours rFVIIa has not been regarded as a routine prophylactic treatment option for FVII deficiency.

Case Reports: We report on our experience of prophylactic treatment with rFVIIa in two FVII deficient patients. Patient one is 43-year-old male with severe FVII deficiency (<1% activity) associated with compound heterozygous Gly78Asp and heterozygous Cys194Tyr mutations. After birth, he experienced severe tissue bleeding complications and later on various joint bleedings with consecutive hemophilic arthropathy. He is now on treatment with rFVII (1.2 mg 3 times per week) since 28 months. Patient two is a 36-year-old male with FVII deficiency of <1% FVII activity. He experienced recurrent mucosal bleeding episodes and large skin hematomas. His genetic defect consists in a homozygous missense mutation (1061 C>T, Ala354Val), a heterozygous missense mutation (218 T>A, Leu73Gln), and a heterozygous frameshift mutation (1391delC, Pro464HisfsX32). The patient is on prophylactic treatment with rFVII (1.2 mg 3 times per week) since 22 months. Since substitution with rFVII, no spontaneous bleeding episodes occurred and no side effects were observed in both patients. In conclusion, prophylactic treatment with rFVIIa in FVII deficient patients appears to be an effective and safe therapeutic option. The mechanisms by which rFVIIa prevents bleeding episodes despite a short half-life of 3 hours remains to be elucidated.
Protein Z in Patients with Newly Diagnosed Multiple Myeloma
Authors: Oleksiuk Jolanta , Kloczko Janusz , Luksza Ewa , Galar Marzenna
Published: April 2020

Background Patients with multiple myeloma (MM) are relatively high risk of developing thromboembolic events. The pathogenesis of prothrombotic coagulation abnormalities in MM is not clear and probably of multifactorial origin. The deficiency of natural anticoagulant mechanisms, acquired resistance to protein C and reduced protein S function, has been described as one of the possible reason of hypercoagulable state in MM patients. Protein Z/Z-dependent protease inhibitor (PZ/ZPI) is a new anticoagulant system has been recently describe. PZ is a vitamin K-dependent protein which serves as cofactor for the ZPI that inactivates activated factor X, XI and IX by different mechanisms. Some reports suggest that protein Z deficiency might imbalance the haemostatic system with thrombotic consequences. However, to the best of our knowledge, no data are available regarding the potential role of protein Z in prothrombotic coagulation abnormalities in MM.

Aim: The aim of our study was to evaluate the plasma concentration of protein Z in relation to disease stage, type and levels monoclonal protein in patients with newly diagnosed untreated multiple myeloma.

Methods: The study population consisted of 41 patients (25 male, 16 female; median age: 63 years) with newly diagnosed untreated multiple myeloma. The presence of monoclonal serum immunoglobulin was found in 33 patients (25 and 8 with IgG and IgA paraprotein, respectively). The disease stage was evaluated according to Durie and Salmon criteria: 4 patients-stage I, 28 patients-stage II and 19 patients-stage III; in 8 of them abnormal renal function (serum creatinine value ≥2.0 mg/dl) was observed. The control group was formed by 19 healhty individuals with age and sex comparable with the patients. Protein Z plasma levels were measured using a commercial enzyme-linked immunosorbent assay (Asserachrom Protein Z; Diagnostica Stago, Asnieres) following the manufacturer’s instruction.

Results: The median protein Z plasma levels in patients with multiple myeloma and control subjects were similar. Median PZ levels were 1.45 ug/ml (range 0.27–3.17 ug/ml) in patients and 1.37ug/ml (range 0.34–1.96 ug/ml) in controls (p≥0.05). No significant correlation was found between protein Z plasma levels and disease stage, monoclonal serum immunoglobulin concentration, and renal function as well.

Conclusions: Our results suggest that protein Z is not an independent risk factor for thrombosis and does not play a significant role in the mechanism of thrombotic complication in patients with multiple myeloma.
Relationship Between Factor VIII Mutation and Inhibitor Development in Previously Treated Patients with Hemophilia a in Korea
Authors: Lee Kun Soo , Lee Soon Yong , Kook Hoon , Hwang Tai Ju , Choi Yong-Mook , Yoo Ki young , Jang In Jin
Published: April 2020

We reported the results of genetic analysis (factor VIII gene, interleukin-10, TNF-alpha) in 54 previously treated patients (PTPs, at least 100 exposure days or 1 year) including 18 with inhibitor history with severe hemophilia A in Korea. We confirmed 20 novel mutations among 35 different factor VIII mutations (intron 22 inversion, missense, deletion, nonsense and splicing), not reported by HAMSTeRS. As our patients were small group, it was not verified that relationship between factor VIII mutation and inhibitor incidence was statistically significant. 9 PTPs with inhibitor history had intron 22 inversion, 7 had missense, 3 had substitution, 1 had nonsense, 1 had small deletion and 1 had splicing. We did not find large deletion. 40 (74.1%) of 54 PTPs were found to have interleukin-10 (IL10) 134 bp microsatellite. IL10 134 bp microsatellite negative group showed statistically higher incidence of inhibitors (odds ratio (OR) 4.0, 95% confidence interval (CI) 1.12 – 14.35; P=0.047). No association was found between inhibitor formation and TNF-alpha gene. Inhibitors were identified in 88.9% of the patients with the TNF-alpha -308 G/G genotype, compared with 11.1% for the TNF-alpha -308 G/A genotype and 0% for the TNF-alpha -308 A/A genotype. Based on this report, prospective study with large sample size may lead to the development of preventive measures to minimize inhibitor formation.
Clinical Analysis of 22 Patients with Primary Fibrinolysis
Authors: Chen Qin-fen , Li Pei , Xie Yi , Chen Bao-An
Published: April 2020

Objective: To analysis the clinical features and therapy of acquired primary fibrinolysis.

Methods: The underlying diseases, clinical manifestations, laboratory findings, treatment and outcomes of 22 primary fibrinolysis were analyzed retrospectively.

Results: 22 patients were enrolled, 13 were male, and 9 were female. The median age was 54.9(17–77) years. The underlying diseases were ulcer of gastric-carcinoma bleeding, colorectal polypectomy by endoscope, intracranial tumor to get worse quickly, metanephric duct galeted by holmium laser+DJ tube implantation, herniorrhaphy of abdominal incision, gaster carcinosectomy, colon carcinosectomy, cerebral trauma and operation of cranium. Patients have bleeding and hemorrhage from surgical incisions or venipuncture or catheter sites in 1 to 7 days after trauma or surgery, with a disproportionately low level of fibrinogen (Fg), high level of tissue type plasminogen activator activity (t-PA) and a relatively normal of thrombin time (PT), activated partial thromboplastin time (APTT), thrombin time(TT), normal or nearly normal of platelet count. Diagnosis of primary fibrinolysis was established clearly, the patients were treated with plasma, cryoprecipitate, fibrinogen and fibrinolytic inhibitor PAMBA. Prognosis was quite good, 18/22 patients were cured.

Conclusion: Primary fibrinolysis may complicate various disorders, most of them are trauma, surgery, and malignancy. Bleeding and hemorrhage from surgical incisions or venipuncture or catheter sites is a predominant clinical feature. Low fibrinogen level with a normal or nearly normal platelet count is a predominant laboratory feature. We should differentiate primary fibrinolysis from the secondary fibrinolysis in DIC. Control of the primary disease, use fibrinolytic inhibitor, and replace depleted clotting factors with plasma, cryoprecipitate, fibrinogen, most of patients can be cured.