bluuebunny/crossref_metadata_2025 huggingface dataset:
Mild Congenital ADAMTS13 Deficiency in a Patient with Familial Recurrent Ischemic Stroke
Authors: Plaimauer Barbara , Skalicky Susanna , Boehm-Weigert Martina , Scharrer Inge , Scheiflinger Friedrich
Published: April 2020

A severe functional deficiency in the ADAMTS13 protease, characteristic for patients suffering from thrombotic thrombocytopenic purpura (TTP), results in persisting prothrombotic ultra-large von Willebrand factor (ULVWF) multimers, which can lead to systemic microvascular thrombosis. The congenital form of TTP is attributed to the presence of homozygous or double heterozygous mutations in the ADAMTS13 gene leading to impaired secretion and/or a functional defect of the protease. Decreased ADAMTS13 activity has been also described for several physiological and pathological conditions. Emerging evidence indicates mild ADAMTS13 deficiency might also increase risk for arterial thrombosis such as for ischemic stroke. We therefore investigated a possible causal relation between moderate ADAMTS13 deficiency and ischemic stroke in one patient selected with the lowest ADAMTS13 activity amongst a cohort of 70 patients with ischemic stroke. At age 34 and 46 years the patient had suffered from idiopathic ischemic strokes with negative common prothrombotic risk factors. We found slightly elevated VWF antigen (161%), ULVWF multimers and reduced ADAMTS13 activity (17%). Genetic analysis identified five nucleotide exchanges in the ADAMTS13 gene leading to one heterozygous missense mutation P353L and four homozygous amino acid polymorphisms (R7W, Q448E, P618A, A732V). Her asymptomatic son presented with normal VWF antigen (99%) and an ADAMTS13 activity of 58% transmitted by a mutant maternal allele and a paternal allele harboring the single Q448E polymorphism. Recombinant expression studies indicated P353L alone causes an impact on ADAMTS13 secretion that is further pronounced in the context of the four polymorphisms. Secreted ADAMTS13 containing P353L or P353L combined with the four polymorphisms presented with similar binding interaction in a binding assay with immobilized multimeric VWF, however, the binding affinity was slightly less than that of wild-type ADAMTS13 or polymorphic rADAMTS13 proteins containing either Q448E alone or all four polymorphisms combined. ADAMTS13 activity analysis suggested that mainly the secretion process or the protein stability rather than the proteolytic activity was affected by the mutations. Co-transfections mimicking the patient’s genotype indicated homozygosity of the four polymorphisms in the patient led to an attenuation of the adverse effect of the heterozygous P353L point mutation resulting in the moderate ADAMTS13 deficiency in the patient. Our results suggest moderate familial ADAMTS13 deficiency might be causally involved in the pathogenesis of ischemic stroke.
Immune Thrombocytopenic Purpura Due to Passenger Donor Lymphocytes in Liver Transplantation
Authors: Mirza Imran , Almugairi Areej , Nahirniak Susan
Published: April 2020

Introduction: Thrombocytopenia is a frequent complication following solid organ transplant but previous reports suggest that the incidence of immune mediated thrombocytopenic purpura (ITP) following liver transplantation is low (0.7%). Although the mechanisms involved in post transplant thrombocytopenia are complex, the presence of alloantibody derived from passenger donor lymphocytes should be considered when severe thrombocytopenia is identified.

Case presentation: Our case is a 58-year-old gentleman who underwent liver transplantation for end stage cirrhosis and hepatocellular carcinoma. His baseline pre-transplant platelet count was low at 83 ×109/L, but he postoperatively worsened to a platelet count of 5×109/L with bleeding sequelae. The liver donor was a 71-year-old previously healthy man who presented with severe thrombocytopenia (platelet count of 3 ×109/L) and hemorrhagic stroke, but no evidence of hematological pathology on bone marrow examination. As the donor history of presumptive ITP was known to the service, antiplatelet antibody assays on both the donor and recipient were performed using the ELISA based GTI-PAK12 kit (GTI Diagnostics, Waukesha, WI). Although there were no detectable antiplatelet antibodies in the organ recipient’s pre-transplant specimens, the immediate post-transplant specimen demonstrated reactivity in all three GPIIb/IIIa wells, corresponding to a stronger but identical reaction pattern in the organ donor. This suggested that the reactivity and subsequent worsening of the thrombocytopenia were secondary to antibodies produced by passenger donor lymphocytes in the transplanted liver. The transplant recipient was treated with corticosteroids, intravenous immune globulin and platelet transfusion with a return to his baseline platelet count and cessation of bleeding symptomatology five days later. Repeat platelet antibody testing in the organ recipient five weeks post-transplant revealed no detectable antiplatelet antibody reactivity, indicating lack of engraftment of the passenger donor lymphocytes.

Conclusion: Passenger donor lymphocytes producing antiplatelet antibodies may be a contributing factor in post-transplant thrombocytopenia. Close monitoring and high clinical suspicion should be present when there is a history of immune/idiopathic thrombocytopenia in the organ donor.
Pregnancy after Allogeneic Hematopoietic Stem Cell Transplantation in Fanconi Anemia Patients: Multi-Institution Cases Report of Female Fertility Recovery
Authors: Nabhan Samir Kanaan , Bittencourt Marco , Duval Michel , Abecasis Manuel , Dufour Carlo , Boudjedir Karim , Rocha Vanderson , Socié Gérard , Passweg Jakob R. , Pasquini Ricardo , Gluckman Eliane
Published: April 2020

Introduction. Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by chromosome instability. Main clinical features include progressive bone marrow failure, skeletal defects, increased susceptibility to malignancy and reduced fertility. Moreover, most recipients of allogeneic hematopoietic stem cell transplantation (HSCT) suffer from secondary infertility owing to gonadal damage from myeloablative conditioning. We report a rare clinical situation of FA patients pregnancy after allogeneic HSCT.

Methods. Retrospective analysis of transplanted FA female patients from 1982 to 2008. Five centers participated in this study on behalf of Aplastic Anaemia Working Party-EBMT. Medical records were reviewed and data collected on a standard case report form including detailed information on diagnosis, transplant procedure, gynecological and obstetrics follow-up.

Results. Among 387 transplanted FA patients we identified 202 females who performed a HSCT with a median age of 10,5 years. Five patients became pregnant after the procedure and one of them, twice. They all had their FA diagnosis confirmed by chromosomal breakage test and a bone marrow aspirate with severe hypoplasia/aplasia. Median age at transplantation was 12 years (range 5–17 years). All patients received myeloablative conditioning regimens (cyclophosphamide with or without thoraco-abdominal irradiation) before a bone marrow transplantation, 4 patients from HLA matched sibling donors and 1 from unrelated donor. During follow-up, 4 patients presented signs of ovarian failure (amenorrhea, low levels of FSH/LH and high levels of estradiol). Apart from 1 patient who spontaneously recovered regular menses, the other three received hormonal replacement therapy (HRT) for this purpose. Pregnancy occurred from 3,5 to 17 years after transplant. One patient had an early interruption with a caesarian section at 27 weeks because of an imminent HELLP syndrome. Other pregnancies were uneventful. Among the newborns, there were no FA positive tests, no congenital anomalies and all of them had normal growth and development. Patients remain alive with a median follow-up of 12 years after transplantation with normal hematological status.

Conclusion. Fertility recovery after HSCT can result from incomplete depletion of the ovarian follicle reserve. HRT should begin promptly to prevent the early and late unwanted effects related to oestrogen deficiency after HSCT. Recovery of normal ovarian function and a viable pregnancy, is a realistic possibility even in Fanconi anemia patients following allogeneic SCT.
Successful Use of Recombinant Factor VIIa (rVIIa, NovoSeven) in Two Patients with Bernard Soulier Syndrome and Dental Extraction
Authors: Zotz Rainer B. , Gerhardt Andrea , Scharf Rudiger E.
Published: April 2020

Background: Bernard Soulier syndrome (BSS) is a platelet function disorder that results from partial to complete deficiency or dysfunction of the GP Ib-IX-V receptor. Specific treatment of bleeding episodes or prophylaxis for the prevention of bleeding during surgical procedures is platelet transfusion. Alloimmunization by HLA antigens and antibody formation to the GPIb-IX-V complex frequently occur and, thus limit future responses to platelet transfusions. Recent patient studies have suggested that rVIIa may be an effective therapy for treatment of bleeding in BSS.

Case Reports: We report on our experience with rFVIIa in two patients with BSS and dental extraction. Patient 1 is a 21-year-old woman (extraction of 2 wisdom teeth). In 2000, after a first extraction of one wisdom tooth, which was covered by administration of DDAVP and platelet transfusion, a bleeding episode occurred, requiring resurgery. Patient 2 is a 14 year old boy (extraction of 4 wisdom teeth). rFVIIa was used as first line therapy. An initial dose of rFVIIa (100 μg/kg/bw) was administered 15 min prior to surgery, followed by three doses of 90 μg/kg/bw at 120 min intervals and three doses at 8 hour intervals postoperatively. In the first three days after surgery, 90μg/kg/bw rFVIIa were administered every 12 hours. Tranexamic acid (CyklokapronÒ 3 × 1 g/d) was started postoperatively and continued for 10 days. There were no postoperative bleeding episodes and no platelet transfusion was required.

In conclusion, rFVIIa in combination with tranexamic acid appears to be an effective and safe therapeutic alternative for prophylaxis of bleeding episodes in dental extraction in patients with BSS.
Evolution of Anti-ADAMTS13 Antibodies in a Refractory Case of Thrombotic Thrombocytopenic Purpura with Fatal Outcome
Authors: Plaimauer Barbara , Knoebl Paul , Skalicky Susanna , Ferrari Silvia , Varadi Katalin , Kolovratova Vera , Turecek Peter L. , Rieger Manfred , Scheiflinger Friedrich
Published: April 2020

Thrombotic thrombocytopenic purpura (TTP) is characterized by systemic microvascular thrombosis leading to life-threatening ischemia of multiple organs, and is associated with a severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13, which permits highly adhesive ultra-large VWF multimers to accumulate in the circulation. Inhibitory and less frequent non-inhibitory anti-ADAMTS13 autoantibodies have been detected in patients suffering from acquired idiopathic TTP. Current treatment with plasma exchange therapy is considered to remove the autoantibodies while concomitantly supplying plasma with the deficient protease. Plasma therapy greatly reduces mortality, nevertheless, still about 10% of the patients die from refractory TTP. To explore the cause of treatment failure in a fatal case of idiopathic acquired TTP we investigated retrospectively the anti-ADAMTS13 immunological profile of a 70-year-old female patient during the course of disease progression. At admittance, she presented with schistocytes, thrombocytopenia and severe neurological disturbances. ADAMTS13 antigen and activity were borderline low in the presence of initially non-inhibiting anti-ADAMTS13 IgG and IgM antibodies, no ADAMTS13 gene aberrations were detected. She had had no previous episodes of TTP. During treatment, the patient received repeated plasma exchanges, supportive extracorporal immunoadsorption and corticosteroid therapy. Despite ongoing treatment anti-ADAMTS13 antibodies still developed and ADAMTS13 activity remained less than 0.1U/ml. After 5 weeks of therapy, during which time the patient was seriously ill with striking neurological limitations, her platelet count dropped and a splenectomy was performed. The patient’s state improved despite a complicated postoperative phase. During the next 4 weeks, her platelet count increased gradually up to 780 G/L, a moderately low ADAMTS13 activity of 0.24U/ml (0.57ng/ml ADAMTS13 antigen) was detected and ADAMTS13-specific antibodies were not measurable. However, within a few days her platelet count dramatically dropped to 20G/L, anti-ADAMTS13 antibodies reappeared and ADAMTS13 activity again was undetectable. The patient’s state deteriorated and she died 2 days later from multiple organ failure. We show the results of the fluctuating anti-ADAMTS13 antibody profile (functional inhibitors and total IgG, IgG subtypes, IgA and IgM) and the pattern of the ADAMTS13 domain-specific reactivity superimposed with the common clinical laboratory data over the patient’s 9-week clinical course.
Burden of Anemia in Cancer Patients Undergoing Chemotherapy: A Retrospective Database Analysis
Authors: Aravind Suresh , Wu Ying , Ranganathan Gayatri , Nalysnyk Luba
Published: April 2020

Background. Along with neutropenia and thrombocytopenia, anemia is a very common hematological adverse effect in patients undergoing chemotherapy. Prolonged and severe anemia is associated with compromised quality of life. The goal of this study was to evaluate the epidemiology of anemia in cancer patients undergoing chemotherapy in USA.

Methods. Pooled data over the period 2000–07 from a large electronic database of 15 community and 3 hospital-based outpatient oncology clinics in the US was analyzed. Adult patients with a cancer diagnosis were grouped as: lung, breast, ovarian, H&N, colorectal, other solid cancers, hematologic and mixed cancers. Chemotherapies used were grouped into platinum, anthracycline, gemcitabine, taxane-based and other regimens. Presence of anemia (defined as Hb<11 g/dL) was evaluated before and during chemotherapy.

Results. 47,159 patients who met protocol specified inclusion criteria were analyzed (mean age 61 (SD 13) yrs, 42% male). The most common tumor types included breast cancer 19.5%, NSCLC 14.9%, colorectal 11.9%, ovarian 3.1% and H&N 2.5%. 75,243 chemotherapy regimens were administered to these patients. Platinum-based regimens accounted for 27.8% of all regimens and were most common among patients with NSCLC (66.6%), ovarian (60.2%), H&N (76.8%), and colorectal (38.9%). Anthracycline-based chemotherapy predominated in breast (46.5%) and hematological cancers (19.1%). Nearly 21% of all cancer patients were anemic at baseline prior to start of chemotherapy. During the course of chemotherapy about 50% of patients experienced anemia. Development of anemia was most common among those receiving gemcitabine regimens (59%). Percentage of patients with severe anemia were: Hb 9–10g/dL = 12–18%, Hb 8–9 g/dL = 5%, and Hb <8 g/dL = 2%. While on chemotherapy, the incidence of anemia was highest among ovarian cancer patients (56.3%), followed by breast (53.3%) and NSCLC (50.9%). Based on data from one large hospital-based site (8,607 patients), the use of RBC transfusions during this period was relatively low (4% in anthracycline to 12% in platinum-based regimens). About 50% of patients received Erythropoietin Stimulating Agents (ESAs) at some point during chemotherapy.

Conclusions. This large community-based analysis observed that nearly 50% of cancer patients undergoing chemotherapy develop anemia. Anemia continues to be a burden in this patient population.
Changes of Intracellular Signal Pathway of mTOR/S6 in T Cells of Low-Risk Myelodysplastic Syndromes
Authors: Zhang Xiang , He Guangsheng , Depei Wu , Zhu Mingqing , Sun Aining
Published: April 2020

Objects: To explore the state of of intracellular signal pathway of mTOR/S6 in FAB low-risk myelodysplastic syndrome-refractory anemia (RA)/RA with ringed sideroblasts (RAS).

Methods: Samples were collected from 18 MDS-RA/RAS patients. The intracellular percentages of p-mTOR, p-S6 and IFN-γ of CD3+, CD3+CD8+ and CD3+CD8− T cells in bone marrow were detected by flow cytometry (FCM). 10 iron deficiency anemia (IDA) patients [male: 3, female: 7, media age: 44 years (from 31 to 72 years)] were determined as case controls and normal controls respectively.

Results

In normal controls, measurement of p-mTOR, p-S6 and IFN-γ in CD3+, CD3+CD8− and CD3+CD8+ T cells were (1.54±1.51)%, (1.94±1.08)%, (2.04±2.03)%; (0.83±0.82)%, (0.91±0.88)%, (0.95±0.93)%; (4.42±3.55)%, (2.35±1.69)%, (4.73±4.43)%, respectively. By 2.62%) of normal controls, MDS group could be divided into two subgroups: MDS-1 with a higher CD3+p-mTOR+ expression, and MDS-2 with a lower expression. In MDS-1, percentages of p-mTOR, p-S6 and IFN-γ in CD3+, CD3+CD8− and CD3+CD8+ T cells were (20.70±18.02)%, (22.94±19.70)%, (26.03±22.91)%; (2.85±2.36)%, (3.36±2.84)%, (4.91±3.88)%; (13.92±12.87)%, (10.05±9.77)%, (16.38±15.49)%; which were higher than normal controls (P<0.05). In MDS-2, the percentages of CD3+p-mTOR+ [ (0.28±0.28)% ], CD3+CD8−p-mTOR+ [ ( 0.46±0.41 )% ], CD3+CD8+p-mTOR+ [ ( 0.47±0.44 )%], CD3+p-S6+[(0.21±0.21)%] and CD3+CD8−p-S6+[(0.17±0.14)%] were lower than those of normal controls (P<0.05) respectively, whlie there were no significant differences in expression of IFN-γ in T cells [ (12.85±12.47)%, (7.11±7.02)%, (20.29±19.84)%] and p-S6 in CD3+CD8+ cells [0.65±0.64)% ] (P>0.05). Upon RAPA treatment, in CD3+, CD3+CD8− and CD3+CD8+ T cells of MDS-1 group, p-mTOR was inhibited more than 50% in 5of 10 cases; p-S6 was inhibited in 2, 4, and 6 cases, respectively, and IFN-γ was inhibited in 4, 4, 3 cases respectively.

Conclusions

In FAB low-risk MDS, the signal pathway of mTOR/S6 was activated, and it was quiescent in normal controls,. The expression of p-mTOR, p-S6 and IFN-γ of this signal pathway in FAB low-risk MDS was heterogeneous. RAPA could suppress mTOR/S6/IFN-γ in partial patients with low-risk MDS.
Non-Myeloablative Allogeneic Hematopoietic Stem Cell Transplantation (NMHCT) for Patients Relapsing after Autologous Stem Cell Transplantation (autoHCT) for B Cell Non-Hodgkin Lymphoma (NHL)
Authors: Freytes Cesar O , Carreras Jeanette , Zhang Mei-Jie , Laport Ginna G. , Van Besien Koen , Lazarus Hillard M. , Hari Parameswaran
Published: April 2020

The prognosis of patients (pts) with NHL who relapse after autoHCT is poor. Myeloablative allogeneic hematopoietic stem cell transplantation has been utilized in these pts since the allograft is tumor free and may induce a graft-versus-tumor effect. Unfortunately, this approach results in prolonged survival for only a minority of pts. NMHCT is increasingly utilized in this setting since it affords the potential benefits of myeloablative transplantation and may reduce morbidity and treatment-related mortality (TRM). Reports using this approach show conflicting results but are limited by small numbers of pts. The purpose of this study was to determine the outcome of pts with NHL who underwent NMHCT after autoHCT failure including TRM, progression/relapse rate, overall survival (OS) and progression free survival (PFS). The study included 267 pts who underwent NMHCT for relapsed B cell NHL after autoHCT failure, from 1997 to 2006 and reported to the Center for International Blood and Marrow Transplant Research. Patients younger than 21, pts who underwent planned second transplants and recipients of cord blood transplants were excluded. The median age at NMHCT was 52 yrs (range 23–70); 64% were male and 61% had a Karnofsky Performance Score (KPS) of ≥ 90%. Fifty six percent of pts had diffuse large cell lymphoma, immunoblastic or follicular large cell lymphoma; 17% had follicular small or mixed NHL and 27% had mantle cell lymphoma. The time interval from 1st to 2nd transplant was < 1 year in 21% of pts, 1 to 2 years in 30% and > 2 years in 49% of pts. At the time of NMHCT, 63% had chemosensitive NHL, 31% resistant NHL and 6% were untreated after autoHCT. Multiple conditioning regimens were used including most frequently melphalan (25%) busulfan (21%) and fludarabine ± cyclophosphamide (26%). A fourth of the pts received 200 cGy of total body irradiation (TBI) as part of the conditioning regimen. The graft source was peripheral blood in 78% and 90% used unrelated donors. Median follow up of survivors was 37 months. TRM at 3 years was 42%. The cumulative incidence of disease progression at 3 years was 36%. The three-year probabilities of OS (figure 1) and PFS were 33% and 22%, respectively. The incidence of grade II-IV acute graft-versus-host disease (AGVHD) at 100 days was 39% and the incidence of chronic GVHD at 3 years was 41%. The most frequent causes of death were progression of NHL (29%), infection (19%), multiorgan failure (19%) and GVHD (14%). Factors associated with a lower risk of NHL progression or death include KPS ≥ 90%, interval between transplants >24 months, TBI as part of the conditioning regimen and complete remission at the time of NMHCT (figure 2). AGVHD decreased the risk of relapse (RR = 0.59, p=0.036)

Figure 1 Figure 1. Figure 2 Figure 2.
Effect of NK Cell on Gvhd in H-2 Haploidentical Bone Marrow Transplantation in Mice
Authors: Zhang Mei , Wu Di , He Pengcheng , Li Jing , Xi Jieying , Xu Hui , Cai Ruibo , Liu Yalin
Published: April 2020

Allogeneic haemopoietic stem-cell transplantation (HSCT) is a definitive cure for many malignant blood diseases. Ideal donors for transplantation are those with completely matched human leukocyte antigen (HLA), kin-relative or no-kin-relative. However only about 25% to 30% people could find HLA matched related-donor and there is only 1/100,000 opportunity to find a matched HLA in unrelated group. Recent years HLA haploidentical transplantation play more and more important role in across the histocompatibility barrier and make about 90% patients possible to get transplantation. However GVHD is still main problem during the transplantation. Recent studies show that natural killer (NK) cells could specially attack recipient antigen-presenting cells (APCs), shown to be responsible for decreasing and improving GVHD. To explore the effect of NK cell on GVHD after H-2 haploidentical bone marrow transplantation(BMT) in mice, we purified NK cells by positive selection with CD49b (DX5) MicroBeads (Miltenyi Biotec product) from donor mice as ingredient in the conditioning regimens, and observed the influence of donor NK cells on GVHD and evaluated the potential role of donor NK cells in protecting against GVHD. Murine model of H-2 haploidentical BMT was established by using Balb/c(H-2d) mouse as recipient, and Balb/c (H-2d)×C57BL/6 (H-2b)(H-2d/b) mouse as donor. Lethally irradiated Balb/c(H-2d) mice were transplanted with Balb/c(H-2d)×C57BL/6(H-2b)(H-2d/b) bone marrow containing donor peripheral T cells and/or NK cells. GVHD and survival rate were studied by observing clinical manifestations and pathological changes. In the group with bone marrow plus T cells, GVHD was induced in 90% mice; but in the group plus with low concentration of NK cells, GVHD was induced in 20% mice; and in the group transplanted with high concentration of NK cells, GVHD was induced only in 10% mice. Compared with the group transplanted only with T cells, the incidences of GVHD in the latter two groups decreased obviously (P<0.01) and the survival rate at 15, 30, 45 and 60 days increased obviously (P<0.01). In mouse H-2 haploidentical BMT, alloreactive NK cells can reduce the incidence of GVHD and increase the survival rate after transplantation in mice.
Ex-Vivo UVA Treated Donor T-Cells for the Induction of Graft Versus Leukemia Effect in Murine Model and Clinical Application
Authors: Samuel Si mcha , Weiss Lola , Hazan Osnat , Resnick Igor B , Shapira Michael Y. , Gesundheit Benjamin , Dray Lilane , Or Reuven
Published: April 2020

Donor lymphocyte infusion (DLI) is a powerful immune-manipulation for the induction of Graft-vs-Leukemia (GVL) effect, but it is associated with high incidence of Graft-vs-host disease (GVHD). Furthermore, the use of DLI across MHC mismatched transplantation is almost infeasible. Extra corporal photochemotherapy for the treatment of GVHD involves extra corporal exposure of peripheral blood mononuclear cells to photoactivated 8-methoxypsorelen (8-MOP) followed by re-infusion of the treated cells. Based on the immunomodulatory effects of 8-MOP and UVA irradiation we investigated ex-vivo similar treatment of donor cells prior to cell therapy in post-transplant murine model. F1 leukemic (BCL1) recipients exposed to total body irradiation followed by bone marrow transplantation (BMT) (C57BL/6→F1). Donor splenocytes incubated with 8-MOP and 10 minuets exposure to UVA irradiation were administrated twice a week for the first 21 days post-BMT. Irradiated hosts inoculated with BCL1 and C57BL/6 cells were examined for the presence of donor cells using fluorescence-activated cell sorting analysis. At 4–6 weeks after BMT chimerism analysis revealed a ratio of 84–91% donor cells. Murine blood cells were tested for Minimal residual disease (MRD) by BCL1- specific PCR for detection of dormant BCL1 at 2 months after BMT. In comparison with the control group, DLI with ex vivo 8-MOP UVA treated splenocytes reduced GVHD and retained GVL effect. In vitro secretion of interferon gamma by treated cells was reduced only by 50%.

Clinical application of this method was performed in 4 high-risk patients after stem cell transplantation from haploidentical related donors:

GVHD T-Cell Dose Day post HSCT Disease UPN - 105/kg +175 Refractory HD+CMV disease #1935 - 104/kg 105/kg +70 +90 Metastatic testicular Ca. #2255 - 5×105/kg 106/kg +84 +127 Refractory neuroblastoma #2316 - 105/kg +130 Relapsed ALL #2323

The potential of Ex-Vivo 8-MOP UVA DLI treated cells should further be investigated for induction of GVL w/o GVHD, to improve chimerism and for anti viral therapy (CMV, EBV and PTLD - Post Transplant Lymphoproliferative Disorders)
Impact of Chemoselective Pressure on Integration Site Patterns of Lentivirally Transduced Human Hematopoietic Stem Cells
Authors: Grund Nadja , Maier Patrick , Appelt Uwe , Allgayer Heike , Wenz Frederik , Zeller W. Jens , Fruehauf Stefan , Laufs Stefanie
Published: April 2020

Hematologic side effects of cancer chemotherapy like myelosuppression are frequently dose-limiting. Lentiviral gene therapy with cytostatic drug resistance gene transfer to human hematopoietic stem cells (CD34+) is a promising approach to overcome this problem. In this context it is of interest if chemotherapy mediated selection has an impact on lentiviral integration site patterns of transduced hematopoietic stem cells (CD34+). Concerning this issue, human CD34+ cells transduced with a lentiviral self-inactivating (SIN) vector encoding MGMTP140K (the O6-BG resistant mutant of O6-methylguanine- DNA methyltransferase) were in vitro treated with the alkylating agent BCNU. For integration site analysis LM-PCR was performed and integration patterns of the treated and untreated CD34+ cells were analyzed and compared with an in silico set of 106 random integrations. We found different integration preferences of the lentiviral vector between either the treated (82 integrations) or the untreated (30 integrations) CD34+ cells and the in silico set: both groups showed

chromosomal preferences, a significant bias for integrations in genes (74,4% in the treated, respectively 70% in the untreated to 40% in the in silico group), especially by favouring introns, a random integration distribution regarding transcription start sites (TSS), and most importantly no significant differences concerning the number of integrations in or near cancer genes.

Concerning all integration characteristics we could not find significant differences when comparing the untreated with the treated group. In conclusion, the general distribution of lentiviral integrations in either untreated or treated human CD34+ cells showed no distinct differences between both groups but significant differences compared to the in silico integration set. These results suggest that chemoselection of cells lentivirally overexpressing a specific chemoresistence gene might not influence the integration pattern. Therefore chemotherapy pressure seems not to hamper the safety of lentiviral vectors in gene transfer studies.
RFUSIN2 - a Clinical Grade Lentiviral Vector Co-Expressing CD80/IL-2 Manufactured Under GMP for a Phase I Clinical Trial Study of Immune Gene Therapy for Poor Prognosis Acute Myeloid Leukaemia
Authors: Chan Lucas , Ingram Wendy , Hardwick Nicola , Darling David , Bhalla Joti , Kizilors Aytug , Kordasti Shahram Y , Mufti Ghulam J , Farzaneh Farzin
Published: April 2020

Patient derived primary AML blasts cells can be efficiently modified (40–100%) to coexpress CD80 and IL-2 following a single round of transduction using a self-inactivating lentiviral vector. This modification produces significant immune stimulation as measured by proliferation, cytokine release and NK and T-cell cytolytic activity against AML in both autologous and allogeneic settings in vitro. In mouse models of leukaemia, CD80/IL-2 modified leukaemic cells result in rejection of previously established leukaemia and the longer survival of the vaccinated animals. Following regulatory approval in the UK, we have initiated a Phase I clinical study using Lentivirus Transduced AML cells expressing CD80 and IL-2 for the potential enhancement of Graft versus Leukaemia effect in poor prognosis AML. The UK regulatory approved vector, RFUSIN2, is a self-inactivating lentivirus vector, which becomes immobile following vector integration. RFUSIN2 encodes CD80 and IL-2 as a fusion protein, which is endogenously cleaved to generate biologically active CD80 that is membrane anchored and IL-2 which is secreted. Here we describe our in-house manufacturing of RFUSIN2 under EU compliant GMP conditions, which resulted in the production of >3x1010 concentrated infectious particles. Extensive safety characterisation of this vector has been carried out. The vector supernatant batch is free of viral contaminants including: Porcine and bovine viruses, other retroviruses, HepA/B/C, HIV-I/II, EBV, and CMV; it is also free of other contaminants including mycoplasma, yeast, fungus and bacteria. RFUSIN2 supernatant is absent of any replication competent lentivirus (RCL) or partial recombinants and is unable to mobilise or to achieve secondary transfer.We present a summary of the protocol for the generation of the autologous AML cell vaccine. The clinical protocol, encompassing the treatment schedule, cohorts, study end points and analytical assays are described.
Correlation Between Glucosylceramide Synthase Gene Polymorphism and Severity Score Index in Type 1 Gaucher Disease
Authors: Alfonso Pilar , Navarro Silvia , Medina Pilar , Irun Pilar , Giraldo Pilar , España Francisco , Pocovi Miguel
Published: April 2020

Background: Gaucher disease (GD), caused by a deficiency of the glucocerebrosidase (GC) enzyme, is characterized by the accumulation of glucosylceramide (GlcCer) within lysosomes, resulting in cellular dysfunction and damage. GlcCer is catalyzed by the glucosylceramide synthase (GCS), also known as UDP-glucose ceramide glucosyltransferase, (UGCG), EC 2.4.1.80, first enzyme in glycosphingolipids biosynthesis. Mutations in the glucocerebrosidase (GBA) gene are required to cause GD, but other factors play an important role.

Aims: GCS gene is a logical candidate to hypothesize that its variability could be involved in clinical severity.

Patients and methods: We have analyzed GCS variants (g.(−295)C>T, g.(−222)ins10, g.148A>G, g.166A>T, g.25510C>G, g.29312A>G and g.34991A>G) in a group of 80 [N370S]+[L444P] heterozygous and 23 N370S homozygous patients.

Results: were related with severity score index (SSI). g.(−295)C>T and g.166A>T SNPs were in complete linkage disequilibrium. In the N370S homozygous, carriers of g.(−222)ins10 have higher SSI than non carriers (6.6 vs 2.4, p<0.004). Patient carriers of the A-allele for SNP g.148A>G had higher SSI than the G-allele carriers (6.7 vs 1.5, p<0.001, for [N370S]+[L444P] heterozygous and 4.3 vs 1.7, p<0.001, for [N370S] homozygous). “In silico” studies and electrophoretic mobility shift assay (EMSA) indicate that 10bp insertion at the promoter region generates a new ETF binding site and the g.148a>g variant at intron 1 changes the affinity for the transcription factor AP-2. GlcCer were elevated in patient carriers of one mutated allele of these two variants.

Conclusion: our results suggest that the genetic GCS variants could influence in the development and progression of GD severity.
Changes in the Atherogenic Profile in Type 1 Gaucher Disease Patients after Substrate Reduction Therapy
Authors: Giraldo Pilar , Puzo Jose , Alfonso Pilar , Irun Pilar , Pocovi Miguel
Published: April 2020

Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid β-glucosidase (GC) leading to accumulation of glucosylceramide within the macrophages of the reticuloendothelial systems. Plasma of GD patients contains low levels of total cholesterol, LDL-cholesterol and HDL-cholesterol. Enzyme replacement therapy (ERT) with imiglucerase and Substrate Reduction Therapy (SRT) with miglustat restores the haemoglobin levels, platelet counts and organomegalies. Previously we have demonstrated that ERT had significant effects on the concentration and metabolism of plasma lipoproteins. However, the effects of SRT on plasma lipid and other atherogenic profiles of GD patients have not been explored. We report the results of long term (36 months) SRT on plasma lipoprotein concentrations in 26 (11 men and 15 women) GD patients. Mean age of patients was 51 (SD ±20.7; range 22–74) years. Ten patients were therapy-naïve to SRT and 16 switched from previous enzyme replacement therapy (ERT). All patients received 100 mg of Miglustat t.i.d with recommendations of a low hydrocarbonate diet in the first weeks. Total cholesterol (TC), triglycerides (TG), low density lipoprotein cholesterol (LDL-c), high density lipoprotein cholesterol (HDL-C), Apolipoproteins (Apo AI, ApoB and Lp(a)), CRP, MPI1b, CCL18 concentrations and chitotriosidase activity were measured before (baseline) and after 12, 24 and 36 months of follow-up. Treatment resulted in a significant increases in HDL-c concentration in naïve patients during the first 24 months (p<0.02) on therapy and it is maintain till 36 months. The same trend was observed in HDL-cholesterol in switch patient but with a less increase that in naïve patients. TC increase slightly in switch (p<0.05) and naïve (p<0.04) patients after 24 and 36 months of treatment respectively. No significant changes in LDL-c during treatment were observed in both switch and naïve patient. In naïve patients, TG, Lp(a) and CRP concentrations, as well as the atherogenic index TC/HDL-c decreased significantly in patients after 24 months therapy and no changes were observed in switch patients. Our results indicate that miglustat treatment have an apparent beneficial effects on plasma lipid, lipoprotein and CRP concentrations resulting in an apparently less atherogenic lipid profile in GD patients. However further studies will be required to determine the impact of miglustat treatment on the risk of coronary heart disease.
Langerhans Cell Histiocytosis Responding to 2-Cda and Imatinib Mesylate (IM)
Authors: Sanchez-Guerrero Sergio , Agreda Gladys , Oviedo Edgar , Lome Carmen
Published: April 2020

BACKGROUND: Langerhans cell histiocytosis (LCH) is a rare condition in which granulomas form in tissues through the accumulation of abnormal histiocytes (Langerhans cells), granulocytes and lymphocytes. Prognosis in patients with disseminated disease has been associated with a chronic course, and a high morbidity and mortality rate. In terms of treatment, etoposide and vinblastine are considered to be the most effective agents in monochemotherapy. Here we report the case of an adult patient with LCH who has improved considerably after treatment with 2-CDA followed by IM.

CLINICAL CASE: 37 year-old Hispanic female with history of burning epigastric pain, nausea and vomiting starting in 1998. In 1999 diarrhea and steatorrhea appeared. Whipple disease was diagnosed in another hospital but treatment was unsuccessful. She came to our hospital in 2003, complaining of the aforementioned symptoms plus severe malnutrition. Upper GI endoscopy and colonoscopy were performed and the biopsies showed infiltration with LCH. Bone marrow (BM) biopsy showed LCH infiltration too. CD117 (KIT) expression was investigated both in gastric and BM biopsies, turning out to be highly positive. Therapies and outcomes are shown in table 1.

DATE DRUGS TREATMENT DURATION WEIGHT (Kg) ALBUMIN (g/dL) GI ENDOSCOPY BIOPSY Dec/03 VBL/VP-16/PDN 5 cycles 33.5 0.99 Polyps and pseudopolyps Infiltrated by LCH. Sep/05 MTX/PDN 18 mos. 39.5 3.18 Polyps and pseudopolyps Infiltrated. Jul/07 2-CDA 9 cycles 55.3 4.0 Polyps Infiltrated. May/08 IM 6 mos. 57 4.0 Nodular pattern. No polyps Decreased infiltration by LCH

CONCLUSIONS: This case illustrates that multisystemic LCH can be successfully treated with, at least two more drug choices: 2-CDA and, in those patients with expression of KIT and/or PDGF, IM.
Risk Index Score for Bacteremia in Febrile Neutropenic Episodes in Children with Malignancies
Authors: Spasova Maria I , Grudeva Janet G. , Kostyanev Stefan S. , Genev Evgeni D. , Stoyanova Angelina A. , Kirina Veselina I. , Moumdjiev Ivan N.
Published: April 2020

BACKGROUND: Nowadays febrile neutropenia is regarded as heterogeneous syndrome and adequate risk stratification of patients as a basis for tailoring empiric antibiotic therapy is attempted. We conducted this prospective study to determine risk factors for bacteremia in febrile neutropenic children that could be assessed in the first days from the onset of fever and based on logistic regression analysis to construct a risk index score for prediction of bacteremia.

MATERIAL AND METHODS: We studied prospectively 199 consecutive episodes of febrile neutropenia for a 4-years period (2000 – 2004), occurring in 80 children with malignancies, treated by conventional chemotherapy. A standardized computer database with a set of variables for each febrile neutropenic episode was used. C-reactive protein was measured at the first fever and depending on its duration - on the 3-rd and 5-th day from the beginning of antibiotic therapy by immunoturbidimetric method. At the onset of fever and before initiation of antibiotic therapy blood cultures were drawn and injected directly into a culture bottle BACTEC Peds plus.

RESULTS: By multivariate logistic regression analysis we determined 5 variables as independent risk factors for bacteremia: the underlying malignant disease (leukemia, NHL-gr.IV), chills, perianal cellulitis, presence of central venous catheter and degree of CRP rise ≥34.5 mg/L between the onset of fever and the 3rd day of empiric antibiotic therapy. This logistic regression model has sensitivity of 93%, specificity of 25%, negative predictive value of 89% and positive predictive value of 36% for the presence of bacteremia (Chi-square: 44, −2 log likelihood:200, Cox & Snell R square: 0.2).

CONCLUSIONS: The present prospective study is characterized by prevalence of hematologic malignancies and relatively high frequency of bacteremia. We identified a low-risk group of 19.1%, comparable to the reported by other groups. Based on the statistical significance of serial measurement of C-reactive protein, which allows definite risk stratification of febrile neutropenic episodes on the 3rd day from the onset of fever, we would recommend sequential empiric antibiotic approach with switch to oral or even outpatient treatment in the low-risk group on the 3rd day.
Study on the Ratio and Function of Macrophages in the Patients with BMMNCCoombs Test(+) Hematocytopenia
Authors: Fu Rong , Wang Yi hao , Shao Zonghong , Chen Jin , Li Lijuan , Liu Hui , Wang Huaquan , Xing Limin , Liu Hong
Published: April 2020

Objective To explore the quantity and function of macrophages in the patients with BMMNC-Coombs Test(+)hematocytopenia.

Methods Sixty-one patients with BMMNCcoombs test IgG(+)hemocytopenia were enrolled in this study. The ratio of macrophages (CD68+/BMMNC), the activation status(CD68+CD69+/CD68+) were detected with Facs. The phagocytic index of macrophage was detected too.

Results There was significant difference of the macrophage ratio and phagocytic index between the patients with BMMNC-IgG(+) and those without BMMNC-IgG(P<0.05);The ratio of macrophage, percentage of activated macrophages and function of macrophages in BM were in kinds of IgG dependent manner. The median value of RBC, Hb and WBC in the patients with CD34 IgG(+) were lower than those in the patients with CD34 IgG(−),but no statistical difference between them was found(P>0.05).The level of WBC in the patients with CD15IgG(+) was significant lower than that of patients with CD15IgG(−)(P<0.05).The levels of RBC, Hb and Reticulocyte of patients with GlycoAIgG(+) was significantly lower than those of the patients with GlycoAIgG(−)(P<0.05).There were significant differences of the levels of RBC, Hb and reticulocyte between the patients with BMMNC-IgG and those without BMMNC-IgG(P<0.05).There were also significant differences of the ratio of macrophage, the percentage of activated macrophages and the function of macrophages between the patients with IgG(+)and those without IgG(P<0.05).

Conclusion The ratio of macrophage, percentage of activated macrophages and function of macrophages were higher in the patients with BMMNC-Coombs test-IgG(+) hematocytopenia. The pathogenic mechanism of this kind of disease might be associated with the activated macrophages phagocyting and destroying BM hematopoietic cells coated with autologous IgG.
Economic Burden of High Risk Acute Lymphoblastic Leukemia (ALL) in Adults and Children
Authors: Newton Nick , McCann Jennifer , Welner Sharon A , Ougari Khalid El
Published: April 2020

Background: Acute lymphoblastic leukemia is a hematological malignancy, characterized by overproduction of immature white blood cells in the bone marrow. Highrisk forms of ALL are typically characterized by poor treatment response, short remission duration, poor survival with conventional chemotherapy and incur high disease-related costs. The most common form of high-risk acute lymphoblastic leukemia in adults is Philadelphia chromosome positive (Ph+) ALL.

Objectives: This study investigated the treatment costs of high-risk ALL from a Canadian perspective.

Methods: Costs for the induction phase of ALL treatment (inpatient costs and length of stay in hospital) were obtained from the Ontario Case Costing Initiative (OCCI) acute inpatient databases. Drug costs for outpatient treatment were obtained from the Ontario Drug Benefit Program and the medical literature. Since no published Canadian guidelines were available treatment pathways and costs for consolidation and maintenance phases of chemotherapy, broken down by age and risk level of disease, were based on the medical literature and expert opinion.

Results: The average total cost of inpatient ALL treatement (induction phase) was $31,694 for both adults and children. The cost for consolidation therapy was $29,244 and $12,753 in adults and children, respectively. The maintenance therapy cost was $7,288 and $3,452 in adults and children, respectively. The high-risk therapy following relapse was $17,100 and $12,000 in adults and children, respectively. The total treatment cost for ALL is estimated at $85,326 in adults and $59,899 in children.

Conclusions: In canada, high-risk ALL exacts a substantial economic burden as a result of rapid disease progression and decreased survival duration. Imatinib (Gleevec®) in Ph+ALL has produced superior results in terms of clinical response, disease-free survival, and overall survival with a good safety and tolerability profile. More comprehensive research is recommended to investigate the economic and humanistic benefits of imatinib as compared to the current therapies in the treatment of Ph+ ALL.
Analysis of Umbilical Cord Blood Stem Cell Therapy for Regenerative Medicine Applications
Authors: Weinthal Joel , Praught Melanie , Brown Heather
Published: April 2020

Over the past 20 years umbilical cord blood (UCB) has become valuable source of hematopoietic stem cells for use in transplantation to treat malignancies, blood disorders and genetic diseases. Cord blood stem cells have shown promising results in animal regenerative medicine studies for the repair of damaged or diseased tissues outside of the hematopoietic lineage. Family UCB banks provide collection and storage services for families to store their child’s UCB for clinical use. This study compared the release of UCB units from one family bank (Cord Blood Registry, CBR) for regenerative medicine versus traditional transplantation since the release of its first UCB unit for regenerative medicine in 2005. UCB was collected from the placenta of consenting mothers and transported to the processing facility from all 50 states and over 80 countries. The UCB was separated into a mononuclear cell fraction from the red blood cells and other non-engrafting cells. The processed UCB is stored in vapor-phase liquid nitrogen at −195° C until requested for use. To date, 230,000 units have been stored. Prior to release, viability and percent CD34+ (or CFU) content were assessed by the treating institution. Through July 2008, a total of 82 UCB units were released for clinical use, 47 of these units (57%) have been used for traditional transplantation and 35 (43%) have been used for regenerative medicine. From January 2005 through July 2008, 47 UCB units were released, 12 (26%) were used in traditional transplantation and 35 (74%) were used in regenerative medicine applications. Over this period the bank experienced a mean of 338% percent growth per year in the release of UCB units for regenerative medicine. The indications for regenerative medicine autologous infusions were cerebral palsy (24 units), brain injury (4 units), type 1 diabetes (6 units), and a rare immune disorder (1 unit). All patients in the regenerative medicine category did not receive any myeloablative chemotherapy or radiation. Released UCB units were stored a mean of 37 months prior to use, the mean CD34+ percent of TNC was 0.53% and mean viability was 95.9%. These data indicate that regenerative medicine applications utilizing autologous cord blood stem cells represent a significant and growing percentage of all treatments facilitated by a family cord blood bank. Regenerative medicine treatments for cerebral palsy and type 1 diabetes have the potential to impact many lives given that 10,000 cases of cerebral palsy and 13,000 cases of type 1 diabetes are diagnosed each year which compares to 2,500 cases of childhood leukemia and 650 cases of neuroblastoma, two common indications for pediatric transplantation. Further analysis is needed to assess the clinical outcomes which will provide background for larger clinical trials in the future.