bluuebunny/crossref_metadata_2025 huggingface dataset:
Autologous Bone Marrow-Derived Stem Cell Therapy: A Promising and Prospective Approach in the Treatment of Patients with Severe Buerger’s Disease.
Authors: Boda Zoltan , Udvardy Miklos , Razso Katalin , Szarvas Mariann , Olah Zsolt , Ilonczai Peter , Toth Judit , Jambor Laszlo , Hunyadi Janos , Sipos Tamas , Kovacs Gabriella , Kappelmayer Janos , Farkas Katalin , Vereb Zoltan , Rajnavolgyi Eva
Published: April 2020

No effective blood-flow enhancement therapies are available for patients with severe peripheral arterial disease (SPAD), thus amputation remains the only option for relief of rest pain or gangrene. Autologous bone marrow-derived stem cell therapy (ABMSCT) is an emerging modality to induce angiogenesis from endothelial progenitors. Eight lower limbs of seven patients with SPAD (seven limbs of six patients with Buerger’s disease) were treated by ABMSCT using isolated CD34+ cells with characterized phenotype and administered by intramuscular injections. The follow-up before and 1-, 3-, 6-, 9- and 12 months after ABMSCT was based on clinical (rest pain, walking distance without pain, non-healing ulcers, ABI) and laboratory (DS-angiography, duplex- and laser ultrasonography, TcPO2) measurements. Therapeutic benefit was demonstrated by complete regression of rest pain in all of the 7 patients, and by the significant improvement of pain-free walking distance (40 m vs 500 m). The average of ABI improved significantly on the treated (before:0.41, after twelve months: 0.83), but did not change on the contralateral limb. Non-healing ischemic ulcers disappeared in five, and became smaller and thinner in two lower limbs, the ulcer remained unchanged in one case, only. The clinical improvement started one month after ABMSCT, it became more prominent after at three months, and the best clinical results were observed after twelve months of the stem cell therapy. In all of the seven cases we observed improvement on the treated limb only, the contra-lateral symptoms and ulcers remained unchanged or worsened. Confirmed by posttrial observations obtained at 18 months after ABMSCT the clinical improvement was evaluated as stable and long lasting. New collaterals were detected by angiography in three patients, but duplex ultrasonography detected improvement in two patients only. Laser ultrasonography did not show significant changes whereas TcPO2 values improved on the foot from 18.80/16.78/23.83 mmHg, and on the calf from 36.66/31.25/45.00 mmHg. These laboratory parameters did not show improvement after 1 month, however, after 6 and 12 months improved values were recorded. Severe adverse events, complications were not observed. We conclude that ABMSCT with isolated CD34+ cells is safe, effective, localized and results in local and sustained clinical benefit for patients with severe forms of Buerger’s disease. We show for the first time that low number of isolated bone marrowderived CD34+ stem cells that involve CD34+CD133+, CD34+CD133−, CD34+CD45− and CD34+CD45+ cells with different vessel forming ability confers vasculogenesis upon intramuscular implantation to patients with Buerger’s disease.
Addition of Rituximab to Mobilization and Conditioning Overcomes the Predictive Value of Follicular Lymphoma International Prognostic Index (FLIPI) Score in Patients with Relapsed FL Undergoing Autologous Stem Cell Transplantation (AUTO).
Authors: Khouri Issa F , Bassett Roland , Samuels Barry , Maadani Farzaneh , Okoroji Grace-Julia , Harrell Robyn , Korbling Martin , de Padua Silva Leandro , Alousi Amin , Hosing Chitra , Giralt Sergio , de Lima Marcos , Anderlini Paolo , Popat Uday , Valverde Rosamar , Hsu Yvonne , Hagemeister Fredrick , McLaughlin Peter , Medeiros L. Jeffrey
Published: April 2020

FLIPI is emerging as an important prognostic factor in patients (pts) with FL. In order to determine its significance in FL pts undergoing AUTO, we examined the outcome of 75 consecutive pts transplanted at the MD Anderson Cancer Center between 02/94 and 04/08. Pts were eligible if they had relapsed chemosensitive disease, and had no HLAidentical sibling donor. Twenty-nine pts were transplanted without rituximab (AUTO-R), and 46 pts received high-dose rituximab (AUTO+R) during stem cell mobilization and on days +1 and + 8 after transplantation as previously described (Khouri, JCO, 2005). Median age (range) at AUTO was 54 (33–76) and 49 (35–63) for the AUTO+R and AUTO-R groups respectively (P =0.002). FLIPI was determined at the time of transplantation; more patients had intermediate-high risk in AUTO+R than in AUTO-R (58% vs 27.5%, respectively, P = 0.011). Other patients characteristics were balanced for gender, time from diagnosis, histology subtypes (grades 1,2, 3a, and 3b), disease stage, LDH, bulk, B-symptoms, B-2microglobulin, bone marrow involvement, number of prior chemotherapy regimens received, remission status (CR vs PR), functional imaging, and co-morbidity score. Median follow-up (range) in months was 20 (1–88) for AUTO+R and 70 (4–167) for AUTO-R. Progression-free survival (PFS) was significantly different between AUTO+R and AUTO-R (P = 0.004), with estimated three-year PFS of 48% for AUTO-R and 79% for AUTO+R. Using Cox proportional hazards regression models, the # of prior chemotherapy regimens received (<3 vs >/=3) (P = 0.015) was the only factor associated with PFS in the AUTO+R group, whereas both age (P=0.010), and risk based on FLIPI (P=0.019) were independently associated with PFS in the AUTO-R group. Pts with low-risk vs. intermediate/high-risk had three-year PFS of 62% and 13%, respectively, in the AUTO-R group, whereas in the AUTO+R group, three-year PFS was 90% and 76% in the low-risk and intermediate/high-risk pts, respectively (Figure).

Conclusions: These results suggest that the addition of R to the mobilization and conditioning improves the outcome in pts with relapsed chemosensitive FL treated by AUTO. The number of prior chemotherapy regimens received rather than FLIPI score is the most important determinant of outcome in this setting.

Figure Figure
Immunogenicity of the Tetravalent Protein-Conjugated Meningococcal Vaccine in Recipients of Related and Unrelated Allogeneic Hematopoietic Cell Transplants (HCT)
Authors: Mahler Michelle , Kernan Nancy A , Boulad Farid , Prockop Susan , Scaradavou Andromachi , Papadopoulos Esperanza B , Abendroth Heidi , Casson Anne , Copeland Catherine , Murphy Maura , Torok-Castanza Joanne , O’Reilly Richard J. , Small Trudy N.
Published: April 2020

Following the successful introduction of the highly immunogenic protein-conjugated Haemophilus influenza and pneumococcal vaccines, Neisseria meningitidis has become the most common cause of bacterial meningitis in the US. Each year, invasive disease (meningitis, sepsis, pneumonia) due to this organism occurs in approximately 500, 000 persons world-wide, including 1400–2800 in the US. Despite rapid institution of appropriate antibiotics, 10%–14% of affected patients die and significant morbidity occurs in up to 20% of survivors. In 2005, the quadrivalent (A, C, Y, W-135) protein conjugated polysaccharide vaccine (MCV4) was approved in the US and recommended for children 11–12 years of age, previously unimmunized adolescents entering college, military recruits, patients with acquired or functional asplenia, and all travelers to hyperendemic or epidemic areas such as sub-Sahara Africa. Improved disease-free survival following HCT has resulted in an increasing number of survivors entering college and traveling abroad. Due to the lack of information on the immunogenicity of this vaccine in transplant survivors, we assessed the responses of 41 patients immunized with MCV4 following an HLA matched related (n=16), mis-matched related (n=2) or unrelated (n=23) HCT. The median age at HCT and at vaccination was 14 years and 18 years, respectively. Patients were transplanted for acute or chronic leukemia (n=28), immunodeficiency disease (n=4), hemoglobinopathy (n=4), HLH (n=1), NHL (n=1), MDS or aplastic anemia (n=3). Fifty percent of patients received an unmodified HCT and four patients were splenectomized prior to vaccination. MCV4 was administered at a median (range) of 3.2 (0.7–12.5) years following transplant. Ninety percent of patients had undetectable titers to all 4 serotypes prior to immunization. Antibody responses were measured by ELISA. Positive response was defined as > 4 ug/ml (serotype A), >5 ug/ml (serotype C), >4 ug/ml (serotype Y), and >3 ug/ml for serotype W-135. Sixty-one percent of patients developed an antibody response to 31 vaccine serotypes, although only 13 developed positive titers to all 4 serotypes. Response to serotypes A, C, Y, and W-135 was observed in 20, 13, 18, and 13 patients, respectively. Responses were significantly poorer in recipients of an unrelated HCT. Of the 7 non-responders who received a second MCV4, 3 of 4 evaluated patients developed titers to all 4 serotypes. This data suggests that although HCT recipients respond to MCV4, the response is sub-optimal, that pre and post vaccine titers should be assessed to ensure adequate protection, and that a booster immunization should be explored in this patient population.
Iron Chelation by Deferoxamine Induces Autophagy
Authors: De Domenico Ivana , Ward Diane McVey , Kushner James P. , Kaplan Jerry
Published: April 2020

Deferoxamine (DFO) is a high affinity Fe (III) chelator produced by Streptomyces pilosus that is used clinically to remove systemic iron in secondary iron overload disorders. DFO cannot be absorbed through the intestine and must be injected. As shown previously, De Domenico et al. EMBO J (2006), expression of Ferroportin (Fpn), the only mammalian iron exporter, can deplete cells of ferritin by lowering cytosolic iron and by exporting iron from cells. Fpn-mediated iron loss induces ferritin degradation by the proteosome. In this study we show that permeable iron chelators, desferirax or deferriprone also induce the proteosomal degradation of ferritin. In contrast, DFO-mediated iron chelation at clinically useful concentrations, leads to ferritin degradation in lysosomes. Immunochemical analysis revealed that DFO-treated cells show increased levels of LC3B, a protein required for autophagy, suggesting that DFO induces autophagy. Treatment of cells with desferasirox or deferriprone did not lead to accumulation of LC3B. Studies using high molecular weight conjugates of DFO or inhibitors of endocytosis showed that the presence of DFO in lysosomes was responsible for the induction of autophagy. Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, does not, however, prevent ferritin loss suggesting there may be an alternate route for ferritin degradation. This hypothesis was confirmed by examining the effect of the proteosome inhibitor, MG132, on DFOinduced autophagy in cells treated with DFO and 3-methyladenine. Addition of MG132 to 3-methyladenine treated cells prevents ferritin degradation. These results indicate that ferritin degradation occurs by two routes: a DFO-induced entry of ferritin into lysosomes and a cytosolic route in which iron is extracted from ferritin prior to degradation by the proteosome.
Long-Term T Cell Immune Reconstitution in Patients Surviving 10 or More Years after Allogeneic Stem Cell Transplantation for Hematologic Malignancies
Authors: Le Quan , Melenhorst J. Joseph , Savani Bipin N. , Hill Brenna , Memon Sarfraz , Shenoy Aarthi , Hensel Nancy F. , Koklanaris Elefteria K , Keyvanfar Keyvan , Hakim Fran , Douek Daniel , Barrett A. John
Published: April 2020

After allogeneic stem cell transplantation (SCT), there is a prolonged immune deficiency and delayed T cell reconstitutions results in significant morbidity and mortality. However limited data are available on immune reconstitution in patients surviving beyond a decade following SCT. Four hundred sixty two patients with hematological malignancies received SCT from an HLA identical sibling in our institute between 1993–2004. Of these, 110 patients 3 or more years post-transplantation, prospectively enrolled in a long-term evaluation protocol. Twenty one of these survived more than 10 years post SCT (median follow-up 11.8 y range 10–14.75y). Diagnoses included chronic myelogenous leukemia (17), acute myelogenous leukemia and myelodysplastic syndrome (3), and multiple myeloma (1). We studied T cell reconstitution in these patients and compared it to samples from their stem cell donors cryopreserved at time of transplant. There was no difference of age at SCT in patients (median age 35.5, range 13–56y) and in the donors (median age 34, range 14–58y). All patients received cyclophosphamide and 13.6 Gy total body irradiation. Patients received T cell depleted bone marrow (n=15) or peripheral blood SCT (n=6) with cyclosporine GVHD prophylaxis and delayed add-back of donor lymphocytes 30–90 days post transplant. Six (29%) developed acute GVHD (grade II–IV) and 18 (86%) chronic GVHD (13 limited, 5 extensive). Six (29%) patients received immunosuppressive therapy (IST) for cGVHD beyond 3 years from SCT but all were off immunosuppressive treatment at the time of study. In the 21 patients there were no significant difference in the absolute lymphocyte, neutrophil or monocyte count, compared with the donor pre-transplant absolute counts of circulating NK and T cell subsets, and B cells were measured using multicolor flow cytometric analysis in 9 patient-donor pairs. Patients had fewer naïve CD4 (p = 0.049) and naïve CD8 (p = 0.004) T cells, fewer CD4 central memory T cells (p = 0.03), fewer CD56 [int] CD16-NKG2A+2D+ NK cells (p = 0.02); and more effector CD8+ T cells (p = 0.04) in patients compared to their donors. ALC and FoxP3+ regulatory T cells were not significantly different between the patients and their donors. The T cell receptor excision circles (TRECs) and T cell receptor repertoire analyses to evaluate thymic function and T cell regeneration is ongoing. In conclusion, patients surviving 10 or more years after allogeneic SCT still show a deficit in the naïve and central memory post-thymic compartment. However these abnormalities appear to be compatible with good health.

Figure Figure
Structural Aspects of Hepcidin-Ferroportin Binding
Authors: Preza Gloria , Fernandes Augustine , Clark Richard J. , Craik David J. , Ganz Tomas , Nemeth Elizabeta
Published: April 2020

Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations in a wide range of organisms, from humans to fish. The hepcidin receptor is the iron channel ferroportin (Fpn), which exports iron from duodenal enterocytes, macrophages and hepatocytes into plasma. Hepcidin binding to Fpn results in internalization and degradation of the ligand-receptor complex and reduced iron efflux from cells into plasma. Abnormal production of hepcidin or abnormal interaction with Fpn causes a spectrum of iron disorders. We analyzed the nature of the interaction and critical structural features of hepcidin and Fpn. The binding of hepcidin to Fpn showed an unusual temperature dependence, with loss of binding/internalization at temperatures lower that 15C. To establish whether initiation of internalization stabilized binding, we used Fpn mutated at Tyr302 and Tyr303, which does not internalize and showed that the mutant was still able to bind 125I-hepcidin, with a similar EC50 and temperature dependence as wt Fpn. We next addressed Fpn structural features required for interaction with hepcidin. Several Fpn mutations in humans cause a phenotype consistent with resistance to hepcidin. We thus generated Fpn plasmids carrying the specific human mutations, transiently transfected cells with the mutants, and measured 125I-hepcidin binding to cells. The results showed that thiol form of Cys326 is essential for hepcidin binding since substitution of this Cys with Ser or Thr preserved the iron exporting function of Fpn but resulted in a complete loss of hepcidin binding, as did the treatment of cells with non-permeable sulfhydryl alkylating agents. The essential role of the C326 residue in hepcidin binding accounts for the early and severe iron overload in patients with C326S or Y substitution. We also showed that the N-terminus of hepcidin is essential for its binding to Fpn. The sequential truncation of five N-terminal residues resulted in a gradual reduction in activity. Ala scanning of the N-terminus showed that Phe4 and Ile6 substitutions resulted in >80% and 50% decrease in binding respectively. To understand the requirements for biological activity at position 4, we tested a series of Phe4 analogues. Substitution with a similar hydrophobic residue, cyclohexylalanine, had no effect on activity; substitution with polar Tyr caused a 50% reduction in activity; substitution with charged residues Lys or Asp resulted in a complete loss of activity, as did substitution with D-Phe. The results indicated that position 4 requires a bulky hydrophobic residue and that the interaction with Fpn is stereospecific. Similarly, substitution of Ile at position 6 with charged residues caused a complete loss of activity. Understanding the molecular framework responsible for hepcidin-Fpn interaction will facilitate the development of drug leads for a range of iron disorders.
Gene Expression Profiling Differentiates Childhood Acute Lymphoblastic Leukemia in Down Syndrome Versus Non-Down Syndrome Patients.
Authors: Rabin Karen R. , Wang Jinhua , Tsimelzon Anna , Morrison Debra , Gaikwad Amos S. , Hogan Laura , Rye Cassia L. , Hilsenbeck Susan G. , Devidas Meenakshi , Heerema Nyla A. , Carroll Andrew J. , Basso Giuseppe , Carroll William L. , Pession Andrea , Bhojwani Deepa
Published: April 2020

Children with Down syndrome (DS) and acute lymphoblastic leukemia (ALL) form a unique biological subset. These patients have generally inferior outcomes in many studies, and an increased incidence of treatment-related toxicities. Cases of DS ALL have a much lower frequency of recurrent prognostically significant chromosomal abnormalities than cases of ALL in the general pediatric population. Global gene expression profiling provides an opportunity to gain insights into pathogenesis and potential therapeutic targets in DS ALL. We performed microarray analysis of RNA from bone marrow samples obtained at diagnosis in 30 DS ALL and 24 non-DS ALL cases using the Affymetrix Human Genome U133 Plus 2.0 array. Unsupervised hierarchical clustering separated cases into two main groups, one of which included 21 of 30 DS samples (Fisher’s exact test, p = 0.013), suggesting inherent biologic similarities. Non-DS samples clustered according to known cytogenetic features. Consistent with recently published data, a subset (13%) of our DS ALL cases were found to have JAK2 mutations. Cases of DS ALL bearing JAK2 mutations did not form a distinct subcluster, suggesting that the JAK2 pathway may be dysregulated via other events in cases of DS ALL with wild-type JAK2. Two-sample comparison of DS versus non-DS ALL cases demonstrated differential expression of 513 genes with p values <0.001 (Figure 1). Oxidative phosphorylation pathway genes were most over-represented among differentially expressed genes, with 35 of 115 genes in this pathway demonstrating down-regulation in DS compared to non-DS ALL (Bonferroni corrected p value < 1 ×10−9), including several cytochrome c oxidase and ubiquinone subunits. Our data indicate that DS ALL blasts may utilize oxidative phosphorylation to a lesser extent than non-DS ALL, a feature which could be exploited therapeutically.

The top 513 genes differentially expressed in DS versus non-DS ALL (Benjamini-Hochberg corrected p values < 0.001) are displayed in a heatmap where genes relatively overexpressed in DS ALL are depicted in yellow, and relatively underexpressed in DS ALL in red. DS ALL cases are indicated by red circles and non-DS ALL cases by white circles. Four DS ALL cases bearing a JAK2 mutation at arginine 683 are indicated by black stars.

Figure 1. Gene expression signature of top differentially expressed genes in Down syndrome (DS) versus non-Down syndrome acute lymphoblastic leukemia (ALL). Figure 1. Gene expression signature of top differentially expressed genes in Down syndrome (DS) versus non-Down syndrome acute lymphoblastic leukemia (ALL).
PIN1 Blocks Granulocytic Differentiation Via C-Jun in Acute Myeloid Leukemia with CEBPA Mutations.
Authors: Pulikkan John Anto , Dengler Viola , Peerzada Abdul , Bohlander Stefan , Tenen Daniel G. , Behre Gerhard
Published: April 2020

Transcription factor CCAAT enhancer binding protein α (C/EBPα) is the master regulator of myeloid differentiation programme. Tumor suppressor function of C/EBPα is shown by the deregulation of C/EBPα in AML by various mechanisms. Mutations in the CEBPA gene are reported in around 9% of patients with acute myeloid leukemia (AML). The CEBPA mutations reported are frame shift mutation at N-terminal domain, which results in C/EBPα-p30 and point mutations at the basic region Leucine zipper. The mutant form of C/EBPα (C/EBPα-p30) displays dominant negative function over the wild type protein and induces AML in mouse model. The mechanism for the dominant negative action of C/EBPα-p30 in AML is poorly understood. Peptidyl-prolyl cis/trans isomerase, PIN1 binds to and isomerizes specific pSer/Thr-Pro motifs. Recent studies show that PIN1 is overexpressed in many types of cancers and it acts as a crucial catalyst for oncogenesis. Here we investigated the role of PIN1 in AML with CEBPA mutations. We report C/EBPα-p30 could induce PIN1 mRNA and protein levels as revealed by quantitative Real-Time RT-PCR analysis and western blot analysis, respectively. Affymetrix mRNA expression analysis shows that PIN1 mRNA is upregulated in patients with AML. Interestingly, the wild type C/EBPα down regulates PIN1 mRNA and protein levels. Chromatin immunoprecipitation assays demonstrate that induction of C/EBPα-p30 results in recruitment of E2F1 in the PIN1 promoter. Silencing PIN1 with inhibitor against PIN1 (PiB) in AML blast cells with CEBPA mutation and in Kasumi-6 cells leads to myeloid differentiation as assessed by myeloid markers by FACS analysis and quantitative Real-Time RT-PCR analysis. Furthermore, PIN1 inhibition by PiB leads to upregulation of wild type C/EBPα protein level. Next, we investigated the mechanism through which the C/EBPα-p30 blocks the wild type protein. c-Jun expression has been shown to be high in AML patients with CEBPA mutation and c-Jun blocks C/EBPα function by proteinprotein interaction. In vivo ubiquitination assay demonstrates that PIN1 increases the stability of the c-Jun protein by inhibiting c-Jun ubiquitination. We also show that c-Jun blocks granulocytic differentiation mediated by C/EBPα as assessed by myeloid specific markers. In summary, C/EBPα-p30 induces PIN1 expression and increases the stability of c-Jun and c-Jun blocks the wild type C/EBPα. Our data suggest the inhibition of PIN1 could be a potential strategy in the treatment of AML patients with CEBPA mutation.
AV513 Promotes Tissue Factor-Mediated Coagulation in Fresh Human Whole Blood from Severe and Moderate Hemophilia a Subjects.
Authors: Powell Jerry Sherman , Knappe Sabine , Harrison Janet A , Patarroyo-White Susannah , Johnson Kirk , Prasad Srinivasa
Published: April 2020

The sulfated polysaccharide extract AV513 accelerates effective clotting of plasma from severe hemophilia A and B subjects, primarily through the extrinsic pathway by inhibiting TFPI activity. AV513 is efficacious in normalizing bleeding times in hemophilia A dogs when administered orally or subcutaneously, either alone or in the presence of very low levels of factor VIII. In this report, we have examined the effects on hemostasis of different concentrations of AV513 (0.25 to 5 ug/mL) added to fresh whole blood from eight severe (FVIII <1 %) and four moderate (FVIII 3–5 %) hemophilia A subjects. Blood was drawn on two separate occasions, 30 days apart, from each subject after receiving no FVIII infusion for 5 days and analyzed using tissue factor-triggered whole blood thromboelastography (TEG) and plasma based thrombin generation assay. In the tissue factor-induced TEG assay, AV513 reduced the whole blood R time (time to initial clot formation) of severe and moderate human hemophilia A blood in a dose dependent manner. The R time values were corrected to the normal range (≤ 27 min) at AV513 concentrations of 0.25 to 1 ug/mL in blood from both severe and moderate hemophilia A subjects. The TEG EC50 (AV513 concentration that reduces R time by 50% from baseline) values were approximately 0.7 ug/mL in both cohorts. Based on two TEG determinations for each subject, the intra-subject baseline variability of whole blood R time had a range of 3–19 % (mean: 10 %) for severe and 2–16 % (mean: 8 %) for moderate subjects. The intersubject baseline R time value varied from 29 to 91 min in the severe group and from 30 to 43 min in the moderate hemophilia A group. Further, an AV513 dose dependent increase in thrombin generation was observed in hemophilia A plasma with maximal thrombin peaks of 52 to 216 nM (mean: 110 nM). For all subjects, we observed a 2–4 fold maximum increase in thrombin peak and a 2–3 fold reduction of both the lag phase time and time to peak thrombin generation.. The EC50 values for peak thrombin generation were 0.2 ± 0.15 ug/mL and 0.7 ± 0.3 ug/mL of AV513 in severe and moderate hemophilia A plasma, respectively. These results demonstrate that AV513 in whole blood from severe and moderate hemophilia A subjects effectively shortened R time in the TEG assay to within the normal range, and that AV513 markedly enhanced thrombin generation in plasma from these subjects. These studies suggest that AV513 may prove useful to promote clotting and prevent spontaneous bleeding in clinical trials for patients with hemophilia A.
Detection of F8 Mutations by High Resolution Melting (HRM) Analysis.
Authors: Bignell Patricia A , Keeling David M. , Giangrande Paul
Published: April 2020

Approximately 50% of severe and all moderate or mild haemophilia A (HA) mutations are single base substitutions, small insertions or deletions with over 900 different mutations having been described. The usual way to screen these HA cases is by DNA sequencing of all 26 exons which is time consuming and expensive.

We have developed the technique of HRM analysis using the Corbett Rotor Gene 6000 for successful high throughput screening of HA mutations. A target sequence was first amplified in the presence of a dsDNA intercalating fluorescent dye followed by in-tube melting analysis. Initially fluorescence is high, but diminishes as the temperature is raised and DNA dissociates into single strands. The observed ‘melting’ behaviour is characteristic of a particular DNA mutation.

In our study we looked at HA mutations in 23 exons,(exons 3–26). Genomic DNA samples from 100 HA patients and carriers were amplified using primers targeting the specific exons. We detected all of the mutations by HRM analysis and each had a distinct ‘melt’ curve which enabled the carriers to be successfully screened. We detected all the HAMSTerS known mutations and our 26 novel mutations in our cohort of patients.

Our results show that HRM could be used as the initial screening method followed by DNA sequencing of the amplicon where a different ‘melt’ curve was observed. It could also be used to rescreen ‘negative’ mutation patients very quickly. This technique is simpler, cheaper and quicker than DNA sequencing method. This method may therefore be particularly useful for prenatal diagnosis and carrier diagnosis.
Molecular Identification of Individual T Cell Clones Specific for Graft- Versus-Leukemia Reaction
Authors: Foltankova Veronika , Matejkova Eva , Bartos Milan , Dendis Milos , Novotna Dana , Mayer Jiri , Michalek Jaroslav
Published: April 2020

Graft-verus-leukemia (GVL) effect in hematopoietic stem cell transplantation (HSCT) is usually complicated by the alloreactivity of donor T cells which leads to acute graft-versus-host (GVH) disease. GVL and GVH reactions are proved to be mediated by different T cell clones. The objective of this study was to identify and characterize T cells clones with specific antileukemia activity without mediating GVHD. We have performed primary mixed leukocyte reaction (MLR) using patient non-leukemic irradiated peripherial blood mononuclear cells (PBMC) as stimulators and donor PBMC as responders. To prepare GVL specific T cells, activated alloreactive T cells were first selectively depleted with an anti-CD25 immunotoxin (Michalek, et al. PNAS2003, 100: 1180–4). Allodepleted T cells were then stimulated in secondary MLR using irradiated leukemia cells from the same patient. Activated leukemia-reactive cells were purified by immunomagnetic selection or by FACS based on INF-γ or CD25 expression, respectively. Clonotypic assay was used for identification of individual leukemia-specific T cell clones (Michalek, et al. Lancet2003, 361: 1183–5; Michalek, et al. J Immunol2007, 178: 6789– 5). This highly sensitive assay is based on detailed analysis of T cell receptor β VDJ unique sequence (TCRB-VDJ). mRNA was extracted from sortred activated cells and cDNA synthetized by anchored reverse transcription. Target TCRB-VDJ gene sequence was amplified by anchor PCR and used to transform bacteria. Bacterial colonies were picked for plasmid isolation and subsequent direct automated sequencing of the TCRBVDJ sequences. We assume that the frequency of particular TCRB-VDJ sequences among bacterial clones after transformation are proportional to the frequency of those sequences in the original population of T cells activated by GVH or GVL reaction. We investigated the presence of individual antileukemic T cell clones in patients with acute myeloid leukemia (AML) and chronic lymphatic leukemia (CLL), and defined them by the TCRB-VDJ unique sequence. The sequences that occured in more than 10% bacterial colonies are likely to represent the most immunodominant clones. Populations of antileukemic T cell clones were oligoclonal, i.e. we observed limited number of individual immunodominat clones which plays important role in GVL reaction. In first CLL patient who had undergone HSCT, six antileukemic T cell clones were identified, four of them are considered to be immunodominant. In second CLL patient after HSCT, only one highly immunodominat autileukemic T cell clone was observed. This specific clone was further monitored by quantitative real-time PCR in patients peripherial blood.
Interleukin-17-Producing T Cells: A New Potential Player Mediating Graft Versus Host Pathology In Patients Undergoing Allogeneic Stem Cell Transplantion
Authors: Dander Erica , Balduzzi Adriana , Zappa Greta , Perseghin Paolo , Lucchini Giovanna , Todisco Elisabetta , Parma Matteo , Biagi Ettore , Di Biasio Iolanda , Villa Antonello , Mina Pamela Della , Migliavacca Maddalena , Longoni Daniela , Rovelli Attilio , Biondi Andrea , D’Amico Giovanna
Published: April 2020

Graft-vs-host disease (GvHD) is a major obstacle to safe allogeneic haematopoietic stem cell transplantation (HSCT), leading to significant morbidity and mortality. Recently, a subset of interleukin (IL)-17-producing cells, named Th17, have been shown to play a central role in the induction of autoimmune-tissue injuries and inflammation. The aim of our study is to analyze the Th17 population in the peripheral blood of transplanted patients experiencing (6) acute, (7) active chronic and (6) chronic GvHD. Both Elispot assay and intracellular staining showed an augmented population of Th17 (both IL-17+/IFN-g− and IL-17+/IFN-g+ producing cells) in patients with both aGVHD and active cGVHD, that can represent up to 2.4% of CD4+ T lymphocytes. In accordance, an increased IL-17 plasma level was detected both in patients with aGVHD (mean IL17A concentration= 14 pg/ml) and active cGVHD (mean IL17A concentration= 12 pg/ml), compared to healthy donors (mean IL17A concentration < 4 pg/ml). Interestingly, the percentage of TH17 cells drastically decreased in patient with quiescent cGVHD. Th17 cells of patients with aGVHD and active cGVHD, were of donor origin, as demonstrated by genotyping, and, in line with what already described, showed high IL23 receptor expression (mean= 40%, range 30%–52%). Noticeably, in the liver and skin GVHD lesions, the solely CD3 cells spontaneously producing cytokines were double positive for INF-γ and IL-17A. Importantly, we demonstrated a strict correlation between the levels of TH17 cells and the clinical status of patients with GVHD. The better understanding of the role played by TH17 in the GVHD pathogenesis could open the way to new highly targeted strategies for the management of GVHD.
A Multicenter, Randomized, Dose Frequency Study of the Safety and Efficacy of Cerezyme® Infusions Every 4 Weeks Versus Every 2 Weeks in the Maintenance Therapy of Patients with Type 1 Gaucher Disease
Authors: Kishnani Priya S
Published: April 2020

Introduction: Cerezyme® (imiglucerase, Genzyme Corporation) is the current standard treatment for type 1 Gaucher disease (GD1). Most patients are infused every 2 weeks. A less frequent infusion schedule at the same total 4-week dose might be equally effective and more convenient.

Objective: To compare the safety and efficacy of two dosing frequencies of Cerezyme for patients with GD1.

Methods: A phase IV, multicenter, randomized 24-month trial enrolled 95 clinically stable adult GD1 patients (mean age 47 years; range 18–82 years) who had received Cerezyme ≥2 years. Patients were randomized to continue their total 4 week dose as 1 infusion every 2 weeks (Q2) or 1 infusion every 4 weeks (Q4). The primary analysis used a composite endpoint of relative change in hemoglobin level, platelet count, liver and spleen volumes, or progression of bone disease and bone crisis. A post-hoc analysis was performed based upon maintenance of therapeutic goals (Pastores et al. Sem Hematol2004; 41(4 Suppl 5):4–14).

Results: Mean 4-week doses (U/kg) of Cerezyme were 70.4±24.9 for Q2 (n=33) and 69.7±21.3 for Q4 (n=62). The primary analysis endpoint was maintained in 80.8% of Q2 and 63.2% of Q4 patients at 24 months. Per the post-hoc analysis, 100% of Q2 and 88.5% of Q4 patients maintained therapeutic goals at 24 months. No Cerezyme-related serious adverse events were reported.

Conclusions: Infusing a total 4-week dose of imiglucerase every 4 weeks appears to be safe and well-tolerated in the majority of patients in this cohort and may be considered for some stable adult GD1 patients who have achieved therapeutic goals on Cerezyme therapy. The dosing frequency of Cerezyme, as with dose itself, must be individualized based upon physician judgment and patient needs as determined by regular and comprehensive monitoring. Long term follow-up of patients on a Q4 dosing regimen is necessary to determine other clinical outcomes, such as bone mineralization and marrow infiltration.
Are Hospitals Following Guidelines for VTE Prevention? the Veneous Thromoembolism Study to Assess the Rate of Thromboprophylaxis
Authors: Amin Alpesh , Spyropoulos Alex , Dobesh Paul , Shorr Andrew , Hussein Mohamed , Mozaffari Essy , Benner Joshua
Published: April 2020

OBJECTIVE: The 7th conference of the American College of Chest Physicians (ACCP7) provides evidence-based guidelines on the type, dose, and duration of thromboprophylaxis in hospitalized patients at risk of venous thromboembolism (VTE), but the extent to which hospitals follow these criteria has not been well studied.

METHODS: Discharge and billing records for patients admitted to any of 16 rural and urban U.S. acute-care hospitals from January 2005 to December 2006 were obtained. Patients 18 years or older who had an inpatient stay 32 days and no apparent contraindications for thromboprophylaxis were grouped into the categories of critical care, surgery and medically ill before being assessed for additional VTE risk factors based on the diagnostic criteria outlined in ACCP7. For patients considered “at risk”, the recommended type (mechanical or pharmacologic), dose, and duration of thromboprophylaxis was identified based on the guidelines and compared to the regimen actually received, if any.

RESULTS: Among the 258,556 hospitalized patients, 68,278 (26.4%) were determined to be at risk of VTE without apparent contraindications for thromboprophylaxis. The proportions of patients who received the appropriate type and dose and duration of thromboprophylaxis were 10.5%, 9.8%, and 17.9% for critical care, medical, and surgical patients, respectively. Of those at risk, 36.8% received no thromboprophylaxis and an additional 50.2% received thromboprophylaxis deemed inappropriate for one or more reasons.

CONCLUSIONS: The implementation of ACCP7 guidelines for type, dosage, and duration of thromboprophylaxis is low in patients at risk of VTE. Fewer than 1 in 7 patients received thromboprophylaxis that met criteria for recommended type, dose and duration. The findings demonstrate a worrisome gap in the performance of evidence-based thromboprophylaxis for hospitalized patients. There is a need for physicians and health systems to improve the levels of awareness and implementation of recommended thromboprophylaxis.
Erythropoiesis Stimulating Agents (ESA) for the Treatment of Chemotherapy Induced Anemia in Patients with Hemoglobin Levels (Hb) <11g/Dl- a Systematic Review and Meta-Analysis
Authors: Paladini Luciano , Clark Otavio , Clark Luciana , Engel Tobias , Faleiros Eneas
Published: April 2020

Background: Anemia is a frequent condition in patients receiving chemotherapy. ESA are effective to control this condition, but a recently published meta-analysis and warnings from regulatory agencies have pointed to a possible increase in mortality in these patients.

Objective: Our aim was to evaluate the safety of ESA when used according to label indications, that is, for patients with chemotherapy induced anemia (rather than cancer induced anemia) with Hb<11g/dl.

Methods: We performed a systematic review and meta-analysis of all randomized controlled trials comparing the use of ESA versus placebo (or no treatment) in patients with chemotherapy induced anemia, for whom ESAs where indicated if Hb dropped below 11g/dl. The primary end-point was mortality. We searched several databases, including MEDLINE, EMBASE, LILACS and CENTRAL, among others. All suitable papers were retrived and data regarding the quality of the studies and mortality rates were extracted. Then, we performed a meta-analysis of these trials, using RevMan 5.0 software.

Results: □There were 17 studies, with 3788 patients, meeting our inclusion criteria. There were 566 deaths among the 2208 patients that received ESA and 488 among the 1580 on control groups. The meta-analysis showed no increase in mortality rates associated with the use of ESA (Relative risk (RR) = 0.95; IC 95% 0.88 to 1.03; P=0.22). Also, there was no heterogeneity in the analysis (I2=0%), showing that the results were consistent among the studies. When we analised the Hb cut-off point in 10g/dl, the meta-analysis results were similar (RR=0.97; IC 95% 0.87 to 1.07; P 0.52; I2=0%).

Conclusion: When used as indicated on label, that is, for patients with chemotherapy induced anemia with Hb<11g/dl ESA are NOT associated with higher mortality rates and remain a safe option for these patients.
Preventing Infections in Cancer Patients: Antibiotics, Hematopoetic Growth Factors or Both? a Network Analysis
Authors: Herbst Christine , Naumann Frauke , Herbst Olaf , Bohlius Julia , Jüni Peter , Engert Andreas
Published: April 2020

Background: Infectious complications such as febrile neutropenia belong to the most serious complications of chemotherapy including stem cell transplantation for cancer. The mortality rate associated with febrile neutropenia is approximately 10%. Meta-analyses have shown that both hematological growth factors (CSFs) and antibiotics prevent infections. A comparison of these two agents or their combination has not been examined in systematic reviews. We therefore conducted a network analysis to determine the role of antibiotics and CSFs for the prophylaxis of infections in cancer patients receiving myelosuppressive chemotherapy or hematopoetic stem cell transplantation (SCT).

Methods: The network analysis included all randomized controlled trials that compared two or more of the following prophylaxis options: no prophylaxis, prophylaxis with antibiotics alone, prophylaxis using CSFs alone or prophylaxis using combinations of both, CSFs and antibiotics. Trails that did report overall survival, on study mortality, infection-related mortality, clinically documented infections, microbiologically documented infections or fever/febrile neutropenia were excluded. A Bayesian network analysis with four treatment options was performed; results are presented as odds ratios (OR) with 95% credibility intervals (CI).

Results: The best prophylaxis in the network with regard to preventing infections and infection-related mortality is the combination of antibiotics and CSFs. Comparing antibiotics alone to CSFs alone yields an OR of 1.00 (95% CI 0.69–1.52) for clinically documented infections. For the combination of both antibiotics and CSFs compared to only antibiotics or only CSFs, the incidence of clinically documented infections was also reduced (OR=0.61, 95% CI (0.46–0.77)). This is also true for the subgroup of patients receiving SCT or patients with acute leukemia.

Conclusions: For patients receiving myelosuppressive chemotherapy, antibiotics and CSFs are similarly efficient in reducing infections. The choice of agent can therefore be based on preferences of the treating physician and the adverse events expected. The best prophylactic regimen however is the prophylaxis using both, antibiotics and CSFs. The combination should be considered for patients at high risk of developing infections such as those receiving SCT or patients with acute leukemia.
Assessment of Nutrition Status in Children with Leukemia: A Study from a Developing Country
Authors: Mukhopadhyay Soma , Dutta Tusi , Dey Sonali , Datta Somnath , Mukhopadhyay Ashis
Published: April 2020

Background: In developing countries 40 % children suffer from mal-nutrition. It has been shown that a good nutritional status can reduce complications of treatment, strengthen the immune system and contribute to the patient’s general well being throughout treatment. A good nutritional status is therefore essential for optimal treatment of a leukemic child. The aim of our study was to see the nutritional status of leukemic children on diagnosis and effect of nutrition on outcome of therapy.

Material & Methods: During period from January 2004 to December 2007 we prospectively analyzed the nutritional status of 400 paediatric patients in Netaji Subhash Chandra Bose Cancer Research Institute, a tertiary cancer center of Eastern India. The age of the patients were 1 to 18 years (median age 12.5 years). The parameter analyzed were weight for age, height for age, total protein, serum albumin and skin fold thickness. The weight for age, height for age and skin fold thickness were taken as normal if they were between 3rd and 97th percentile curve of the growth chart recommended by the Indian Council of Medical Research. The albumin level and the total protein was considered normal if the value is equal to or more than 3gm% and 5.8gm% respectively.

Result: It was seen that total 56 patients (14% children) were low weight for age, 40 patients (10% children) were low height and 67 patients (16.75% children) had low mid arm circumference. Total 44 patients (11% children) had low serum albumin while 80 patients (20% children) had low serum protein. It was found that low weight for age, low serum albumin and low mid arm circumference were significance factors in remission induction, disease free survival and toxicity of chemotherapy (p < 0.005).

Conclusion: We conclude that mal-nutrition is a major finding in cancer patients with leukemia in developing country like ours. The patient with mal-nutrition had less remission in induction, disease free survival and more toxicities during therapy as compared to well-nourished children.
A Psychometric Analysis of Parents of Children with Leukemia
Authors: Zheng Huyong , Wu Xinyi , Zhang Ruidong , Huo Liqin , Liu Huaqing , Wang Xiumei , Hu Yamei , Lu Xiaoyan , Gao Huifang
Published: April 2020

To investigate the anxiety and depression of parents whose children suffered from leukemia and to develop a set of theoretical guidance for later psychologic intervention, parents of leukemia children treated in Beijing Children’s Hospital (BCH) from March of 2004 to March of 2006 were evaluated with Family Environment Scale (FES) and Hospital Anxiety and Depression (HAD) questionnaires. They were tested in three phases relating to three groups: parents of newly-diagnosed-children group, parents with or without psychological intervention groups. Parents in intervention group were given individual psychotherapy, group therapy or systemic family therapy. A total of 353 questionnaires were analyzed, including 138 in newly-diagnosed-children group, 103 in intervention group and 112 in non-intervention group. Results showed:

The family environment scores of expressiveness, independence, achievement, cultural, recreational orientations and organization were significantly lower, but the scores of conflict, ethics religion and control were much higher in parents of newly-diagnosed-children group than the normative population. For example, the scores of expressiveness and conflict were 4.9 and 2.3 in normative population, but the scores were 2.3 and 3.9 in parents of leukemia children. The HAD scores of parents of newly-diagnosed-children group showed that the scores of anxiety and depression were 38.4% and 24.1% higher than the baseline value respectively. The scores of parents’ cultural, recreational orientations and independence increased to normal level in six months after their children received treatment; Fathers’ conflict score decreased to normal level in intervention group (3.0) compared to non-intervention group (4.4). The scores of parents’ anxiety and depression reduced to baseline six months after their children received treatment.

We concluded that the psychologic distress of parents was influenced tremendously by their leukemia children. At the beginning of diagnosis their anxiety, depression and conflict were significantly elevated. Most parents could cope with their leukemia children by timely and effective psychological supports. Professional psychological intervention is most helpful in reducing family conflicts.
Cdk1-Dependent Phosphorylation ofNPM Overrides G2/M Checkpoint and Increases Leukemic Blasts in Mice
Authors: Du Wei , Zhou Yun , Pike Suzette , Pang Qishen
Published: April 2020

An elevated level of nucleophosmin (NPM) is often found in actively proliferative cells including human tumors. To identify the regulatory role for NPM phosphorylation in proliferation and cell cycle control, a series of mutants targeting the consensus cyclin-dependent kinase (CKD) phosphorylation sites was created to mimic or abrogate either single-site or multi-site phosphorylation. Cells expressing the phosphomimetic NPM mutants showed enhanced proliferation and G2/M cell-cycle transition; whereas nonphosphorylatable mutants induced G2/M cell-cycle arrest. Simultaneous inactivation of two CKD phosphorylation sites at Ser10 and Ser70 (S10A/S70A, NPM-AA) induced phosphorylation of Cdk1 at Tyr15 (Cdc2Tyr15) and increased cytoplasmic accumulation of Cdc25C. Strikingly, stress-induced Cdk1Tyr15 and Cdc25C sequestration were completely suppressed by expression of a double phosphomimetic NPM mutant (S10E/S70E, NPM-EE). Further analysis revealed that phosphorylation of NPM at both Ser10 and Ser70 sites were required for proper interaction between Cdk1 and Cdc25C in mitotic cells. Moreover, the NPM-EE mutant directly bound to Cdc25C and prevented phosphorylation of Cdc25C at Ser216 during mitosis. Finally, NPM-EE overrided stress-induced G2/M arrest, increased peripheral-blood blasts and splenomegaly in a NOD/SCID xenograft model, and promoted leukemia development in Fanconi mouse hematopoietic stem/progenitor cells. Thus, these findings reveal a novel function of NPM on regulation of cell-cycle progression, in which Cdk1-dependent phosphorylation of NPM controls cell-cycle progression at G2/M transition through modulation of Cdc25C activity.