bluuebunny/crossref_metadata_2025 huggingface dataset:
PR1 Vaccine Elicited Immunological Response after Hematopoietic Stem Cell Transplantation Is Associated with Better Clinical Response and Event-Free Survival.
Authors: Qazilbash Muzaffar H. , Wieder Eric D. , Thall Peter F. , Wang Xuemei , Rios Rosa L. , Lu Sijie , Kant Shreya , Giralt Sergio A. , Estey Elihu H. , Cortes Jorge , Komanduri Krishna , Champlin Richard E. , Molldrem Jeffrey J.
Published: October 2019

Background: PR1 peptide has been established as a human myeloid leukemia-associated antigen. We studied PR1 peptide vaccine in a phase I/II clinical trial in HLA-A2 + patients with AML, MDS and CML. To address whether prior HSCT or prior use immunosuppressive drugs would prevent PR1-induce cytotoxic T lymphocyte (PR1-CTL) immunity after vaccination with PR1 peptide vaccine, we studied the outcome in 20 patients with a prior HSCT, who were treated on the PR1 vaccine trial.

Methods: Twenty patients (13 with AML or MDS, 7 with CML) were vaccinated at a median time of 9.5 months (range: 1–220) after HSCT. Sixteen patients had received a prior allogeneic HSCT (12 had allogeneic related, 3 had allogeneic unrelated, and 1 had syngeneic) and 4 patients had a prior autologous HSCT. At the time of vaccination, 5 patients were in CR, and 15 had measurable disease. Patients could not receive systemic immunosuppressive therapy for at least 4 weeks prior to vaccination, and had to be free of acute or chronic GVHD requiring systemic therapy. The vaccine was given subcutaneously every 3 weeks for a total of 3 injections at one of three dose levels: 0.25, 0.5 and 1.0 mg. GM-CSF at a dose of 75 mg was injected subcutaneously into the same site. Immune response to the vaccine (IRV) was defined as > 2-fold increase in PR1-CTL by PR1/HLA-A2 tetramer assay.

Results: After a median follow up of 56.5 months (range: 27–89), toxicity was limited to grade I/II injection site reactions in 7 (35%) patients. IRV were observed in 11/20 (55%) patients. Nine of 11 (82%) IRV+ patients versus 1 of 9 (11%) IRV- patients had clinical responses (p = .005). Median event-free survival (EFS) was 23.8 months in IRV+ patients versus 1.9 months in IRV- patients (p=0.03). Median overall survival (OS) in IRV+ patients has not yet been reached vs. 40 months in IRV- patients (p = 0.08). Only 2 patients with pre-existing, limited chronic GVHD experienced a mild exacerbation within 3 months of vaccination, which was controlled with topical steroids. PR1-CTL were enriched in central memory phenotype (TCM) that persisted up to 4 years in clinical responders. Univariate and multivariate Cox proportional hazards analyses showed a low pre-vaccine bone marrow blast count (<10%) was associated with a lower risk of progression (p=0.001 and 0.001, respectively). Type of HSCT, interval between HSCT and PR1 vaccine, PR1 dose level and disease status at HSCT did not have a significant impact on EFS or OS.

Conclusion: PR1 vaccine produced PR1-CTL in 11/20 (55%) patients after HSCT. IRV was associated with significantly better clinical response and longer EFS.

Figure Figure
Minimal Residual Disease Detected Prior to Transplantation Is Associated with Adverse Outcome in Pediatric Acute Lymphoblastic Leukemia.
Authors: Foster Jennifer H , Woolfrey Anne , Wood Brent , Thomson Blythe
Published: October 2019

Background: Acute lymphoblastic leukemia (ALL) is the most common form of malignancy in children. Advances in treatments have made ALL the disease highly curable; however for those patients who relapse, hematopoeitic stem cell transplantation (HSCT) offers a reasonable chance of cure. Minimal residual disease (MRD) detection by Multiparametric Flow Cytometery (MPF) is being used for risk adapted treatment decisions in many ALL trials. We present a series of 31 pediatric ALL patients who had morphologic and MPF evaluation of disease burden prior to ablative HSCT.

Methods: Thirty one patients were treated at Children’s Hospital and Regional Medical Center, Seattle, WA for relapsed or very high risk ALL, were in complete morphologic remission, and received an ablative HSCT from May 2006-May 2008. Twelve patients were in second or third complete remission (CR) and 19 were in first CR. Eleven patients received a matched related donor, 20 patients received a unrelated donor graft. All patients underwent marrow evaluation including morphology and MPF within four weeks of their transplant date. The MPF was done by 7 or 9 color flow cytometry using the following reagents for B lineage: CD10, CD19, CD20, CD34, CD38, CD58 and CD45 and for T lineage: CD2, CD3, CD4, CD5, CD7, CD8, CD34, CD56, and CD45. Transplant regimens were total body irradiation-based (1320 cGy) with either cyclophosphamide (n=24) or fludarabine (n=6). MRD+ was any detectable leukemia >0.01% of cells. All patients were in morphologic remission (< 5% blasts) at time of transplant. Events were defined as relapse or deaths.

Results: 21 patients were MRD-, 10 were MRD+. The 2 year event free survival (EFS) for the entire group was 56% (+/−22%). The EFS at 20 months for those patients in CR1 and CR2/3 were 62% (+/−32%) and 40% (+/−32%), respectively. EFS, relapse risk and non relapse mortality was analyzed with respect to MRD status:

MRD+ (n=10) MRD- (n=21) p value EFS 36% (+/−32%) 68% (+/−26%) 0.037 Relapse Risk 48% (+/−36%) 13% (+/−16%) 0.036 Non-relapse Mortality 30% (+/−36%) 23% (+/−26%) 0.45

Discussion: We present a single institution series of patients treated for high risk or relapsed ALL who underwent disease evaluation prior to HSCT with MPF. With the small number of patients evaluated, it appears that any amount of disease detected by MPF was an adverse risk factor for recurrence. Those patients who were MRD+ experienced a higher risk of death from relapse, however, experienced no difference in non-relapse mortality. Resistant disease as detected by MRD analysis at time of transplant is a marker for poor outcome.
Allogeneic Stem Cell Transplantation for Patients with Chemo-Refractory or Progressive Aggressive Non-Hodgkin’s Lymphomas.
Authors: Hamadani Mehdi , Blum Kristie A. , Elder Patrick , Lin Thomas S , Porcu Pierluigi , Hofmeister Craig C. , Blum William , Marcucci Guido , Benson Don , Devine Steven
Published: October 2019

Background: Patients (pts) with chemo-refractory (Cref) and/or progressive aggressive non-Hodgkin’s lymphomas (A-NHL) generally have a poor clinical outcome. Autologous stem cell transplantation (SCT) has limited efficacy in this setting. Studies reporting outcomes of allogeneic (Allo) SCT in pts with Cref A-NHL are limited.

Methods: 108 pts with A-NHL underwent Allo-SCT between 1988 and 2007 at our institution. 46 A-NHL pts with Cref (i.e pts with <50% reduction in tumor size and/or persistent bone marrow involvement following chemotherapy) or progressive disease (PD) by CT-criteria were eligible for this analysis. Positron emission tomography (PET) or PET/CT scans were not performed to assess response.

Results: The median age was 46 years (range 22–63 yrs). 39 pts received matched sibling allografts, while 7 underwent unrelated donor SCT. All except 3 pts received myeloablative conditioning. 38 pts received BU/CY-based conditioning, while BEAM (n=5) or Flu/Bu (n=3) was employed in the remaining pts. Diagnosis included diffuse large B-cell lymphoma (n=18), Burkitt’s lymphoma (n=3), transformed lymphoma (n=5), mantle cell lymphoma (n=11) and T-cell lymphomas (n=9). The median number of prior therapies was 3 (range 1–8). 32 pts had Cref disease, while 14 had PD. 41 pts had stage III/IV disease, 23 had elevated LDH, while 36 had extranodal involvement. Median follow-up of surviving pts following Allo-SCT is 5-yrs. The 5-yr overall survival (OS), progression free survival (PFS), and relapse rate for the whole cohort (n=46) was 38%, 34%, and 35% respectively. Rate of grade II-IV acute graft-versus-host disease (GVHD) was 43% (n=20). Among the 33 evaluable pts rate of chronic GVHD was 75%. Overall non-relapse mortality rate was 37%. No significant difference in the baseline characteristics of pts with Cref and PD was present. The 5-yr OS and PFS rates for pts with Cref and PD were 46% vs. 21% (p-value=0.01; log-rank test), and 46% vs. 7% (p-value=0.0002; log-rank test) respectively. On multivariate analysis only PD at the time of SCT predicted for worse OS and PFS.

Conclusion: Our study shows encouraging efficacy of Allo-SCT in a group of A-NHL pts with Cref disease by CT scan-criteria who often fail following Auto-SCT. However outcomes of pts with PD remain dismal despite Allo-SCT, and our data question the practice to perform allografting in A-NHL pts with PD. We hypothesize that PET scans may help better define patients with aggressive NHL appearing not to have responded to salvage chemotherapy by standard CT criteria that still may derive significant benefit from allografting.
Haploidentical Hematopoietic Stem Cell Transplantation in Children Leukemia and Lymphoma with G-CSF Mobilized Marrow Grafts without T Cell Depletion: A Single Center Report of 45 Cases.
Authors: Wang HengXiang , Yan Hong-Min , Duan Lian-Ning , Zhu Ling , Xue Mei , Liu Jing
Published: October 2019

Haploidentical hematopoietic stem cell transplantation (Haplo-SCT) has been increasingly used in the treatment of patients with hematopoietic malignancies without immediate HLA-matched donors. Here, we describe a protocol for the management of hematopoietic malignances in children and adolescents who received T cell replete G-CSF-mobilized bone marrow grafts from HLA-mismatched parents. The donors were administered G-CSF for 7 consecutive days before marrow grafts containing a medium nucleated cells of 8.1×108/kg (range: 6.1–11.5×108/kg) were collected. 45 patients were pre-conditioned with high-doses of cytarabine and cyclophosphamide plus total body irradiation or BU for chronic myeloid leukemia patients. The regimen for acute graft versus host disease (aGvHD) prophylaxis included antithymocyte globulin, monoclonal antibody against CD25 (Simulect), CsA, MTX and mycophenolate mofetil. Three of forty-five patients died of engraftment failure, multiple organ failure and TTP, others achieved complete donor chimerism with the medium days for neutrophil (>0.5′109/L) and platelet recovery (>20′109/L) were 17(12–23 days) and 19(12–27days). Of the 42 evaluable patients, 3 cases developed grade III/IV aGvHD, and 9 of 32 cases who survived longer than 3 months developed chronic GvHD. Twenty-one patients died within a medium follow-up of 36 months(24–63 months), including 2 of transplantation related death, 11 of leukemia relapse, 4 of CMV and fungal infections, and 2 of aGvHD and 2 cases died of other reasons. The disease-free survival rate was 53%. Multivariate analysis demonstrated that the overall survival was not significantly reduced in refractory leukemia and seemed not associated with the mismatch numbers of HLA loci. These results indicate that T cell replete SCT with mobilized bone marrow from HLA-2 or -3-antigen mismatched parents could be an optional strategy for children with hematopoietic malignancies who lack immediate access to a HLA-matched stem cell source.
Epigenetic Regulation of the WNT Canonical Pathway in Mantle Cell Lymphoma.
Authors: Peters Anthea C , Chung Randy , Wong Charles S , Young Leah C , Reiman Tony , Lai Raymond
Published: October 2019

Dysregulation of the WNT signalling pathway is an important event in the pathogenesis of several types of cancer, including colon cancer and acute leukemia. When a WNT protein binds to its receptor, glycogen synthase kinase (GSK)-3β is inactivated by phosphorylation (i.e. pGSK-3β) allowing β-catenin to accumulate in the nucleus and promote transcription of proliferative genes. Conversely, a protein complex that includes GSK-3β initiates destruction of β-catenin if no WNT protein binds its receptor or if an antagonist interferes with WNT ligand-receptor interaction. We previously demonstrated that the WNT canonical pathway is often constitutively active in mantle cell lymphoma (MCL), a subtype of B-cell non-Hodgkin lymphoma. We showed that GSK-3β is phosphorylated, thus inactive, in two-thirds of MCL cases, and that a high level of pGSK-3β correlates with both β-catenin expression and poor patient survival. In order to elucidate the mechanisms of constitutive WNT signalling in MCL, we investigated the RNA expression of six WNT pathway antagonists including WIF-1 using fresh tumour samples and three types of cultured MCL cells. In vivo WIF-1 expression was abundant only in tumours shown to lack pGSK-3β expression. We also examined the regulation of these WNT antagonists by promoter methylation by treating cultured cells with demethylating agent 5-aza-2’-deoxycytidine. The WNT pathway inhibitors were often negatively regulated in MCL-derived cell lines by epigenetic DNA methylation; inhibitors that were not expressed at baseline showed expression after demethylation treatment in at least one MCL cell line. Methylation-specific PCR confirmed that the promoter region of WIF-1 was methylated in almost half of MCL tumour samples. Our data demonstrates that in MCL several antagonists of the canonical WNT pathway are negatively regulated by promoter methylation, thereby allowing aberrant WNT pathway activation. This data confirms that WNT signalling is important in MCL pathogenesis, and identifies potential therapeutic targets for MCL.
Synergistic Pre-Clinical Activity of Combined Epigenetic Therapy with the Novel Histone Methyltransferase EZH2 Inhibitor 3-Deazaneplanocin and Histone Deacetylase Inhibitor Panobinostat against Human AML Cells.
Authors: Fiskus Warren , Wang Yongchao , Jillella Anand , Ustun Celalettin , Johnston Pace , Joshi Rajeshree , Rao Rekha , Ataja Peter , Marquez Victor , Bhalla Kapil N.
Published: October 2019

Lysine specific histone methylation and deacetylation and DNA hypermethylation are involved in the epigenetic silencing of tumor suppressor genes (TSG), e.g., p15 and p16. The multi-protein complex PRC (polycomb repressive complex) 2 that contains the three core proteins EZH2, SUZ12 and EED, has intrinsic histone methyltransferase (HMTase) activity. This is mediated by the SET domain of EZH2, which induces tri-methylation (3Me) of lysine (K)-27 on histone H3, regulates the expression of HOX genes and promotes cell proliferation and aggressiveness of neoplastic cells. In the present studies we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-Deazaneplanocin A (DZNep) dose-dependently (200 nM to 2.0 uM) depletes EZH2, SUZ12 and EED levels, inhibits 3Me K27 on H3 while inducing K27 H3 acetylation in the cultured human AML HL60 and OCI-AML3 cells and in primary, patient-derived AML blasts. DZNep treatment also induced the levels of p16, p21, p27 and FBXO32 while depleting cyclin E and HOXA9 levels. Treatment with DZNep induced PARP cleavage activity of the caspases and apoptosis in the cultured and primary AML cells. DZNep promoted proteasomal degradation of EZH2 and SUZ12, since co-treatment with bortezomib significantly restored EZH2 and SUZ12 levels in the AML cells. We had previously reported that treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) (LBH589, Novartis Pharmaceutical Corp) also depletes the levels of EZH2, SUZ12 and EED in cultured and primary AML cells (Mol Cancer Ther.2006; 5:3096). Within the PRC2 complex, EZH2 bound and recruited the DNA methyltransferases DNMT1, and treatment with PS also disrupted the interaction of EZH2 with DNMT1, attenuated DNMT1 levels and its binding to the EZH2-targeted gene promoters, e.g., p16 and JunB. Here, we also demonstrate that, as compared to treatment with either agent alone, co-treatment with DZNep and PS caused more depletion of EZH2, SUZ12 and EED, more induction of p16, p21 and p27, as well as synergistically induced apoptosis of AML cells (combination indices < 1.0). Additionally, DZNep induced apoptosis of HL-60/LR cells that are resistant to HDACs including PS, as well as sensitized HL-60/LR cells to PS. Taken together, these findings indicate that targeting EZH2 and the PRC2 complex is an effective epigenetic therapy of AML that also overcomes resistance to HDAC inhibitors. Additionally, combined epigenetic therapy with DZNep and PS exerts synergistic in vitro activity against human AML cells, suggesting that this combination may be a promising novel treatment for AML.
Haploinsufficiency of Ebf1 Collaborates with BCR-ABL1 to Decrease the Latency of Acute Lymphoblastic Leukemia.
Authors: Goorha Salil , Lenny Noel T. , Miller Christopher B , Perry S. Scott , Su Xiaoping , Williams Richard T , Mullighan Charles G , Downing James R.
Published: October 2019

In previously published genome-wide copy number analysis of leukemic samples from 242 pediatric acute lymphoblastic leukemia (ALL) patients, we reported that mutations in genes regulating B lymphoid development are the most common lesion in B-progenitor ALL, and these include PAX5, IKZF1, and EBF1. Mono-allelic deletion of EBF1 was observed in 8/200 B-progenitor leukemia samples, including a BCR-ABL1 ALL. EBF1 encodes a transcription factor that is required for the development of B cells, and with E2A regulates the expression of B-lineage specific genes. Mice null for Ebf1 arrest B cell development at the pro-B cell stage, whereas Ebf1+/− mice have a 50% reduction in the number of immature and mature B cells but a normal number of pro-B cells. Importantly, neither haploinsufficiency nor the complete loss of Ebf1 results in the development of leukemia in mice. To examine the role of genetic alterations targeting B-lymphoid differentiation in the pathogenesis in BCR-ABL1 ALL, we transduced Ebf1+/+ and Ebf1+/− bone marrow cells with MSCV-GFP-IRES-p185 BCR-ABL1 retrovirus and transplanted the resultant cells into lethally irradiated wild-type C57BL6 recipient mice. Mice transplanted with BCR-ABL1 Ebf1+/− cells developed B lineage ALLs at a shorter latency than observed with BCR-ABL1 Ebf1+/+ cells (median overall survival of 17 days in Ebf1+/− vs 42 days in Ebf1+/+, P<0.0001). All leukemias had a B220+Cd19+Bp1+ pre-B cell immunophenotype; however, the leukemias that developed from the Ebf1+/− cells aberrantly expressed high levels of the stem cell marker Sca1 (mean fluorescence level for Sca1 of 69.6 in Ebf1+/− (n=22) vs 16.8 in Ebf1+/+ (n=14), p<0.0001). To begin to understand how a decrease in the copy number of Ebf1 may contribute to leukemogenesis, we examined early B cell development in bone marrow (BM) cells from two week-old C57BL6 Ebf1+/− and Ebf1+/+ mice. Our analysis confirmed previous reports indicating a 2-fold reduction of B220+CD43− B cells in Ebf1+/− compared to Ebf1+/+ mice. Interestingly, however, we also detected an approximately 6-fold increase in a transitional population of B220loIL-7R+cKitlo Pre-pro B cells that also expressed Sca1 (2194 mean number of Ebf1+/− cells per 100,000 BM cells (n=10) vs 372 mean number of Ebf1+/+ cells per 100,000 BM cells (n=8), p<0.0001), an observation that raises the possibility that Ebf1 haploinsufficiency expands the pool of cells that are susceptible to transformation by BCR-ABL expression. It will be important to examine whether the accelerated tumorigenesis resulting from Ebf1 haploinsufficiency is a consequence of a subtle shift in differentiation, or some alternative mechanism of oncogenic cooperativity. Studies are underway to directly assess the role of B220loIL-7R+cKitlo Sca1+ cells in BCR-ABL1 driven ALL.
ADVATE [Antihemophilic Factor (Recombinant), Plasma/Albumin- Free Method] Post-Authorization Safety Surveillance (PASS): Safety and Efficacy Following Product Switch in Routine Clinical Practice.
Authors: Luu Huong , Spotts Gerald , Gajek Hartwig , Kriukov Alex , Berg Roger , Stephens Darby , Barker Kerry , Ewenstein Bruce M.
Published: October 2019

A prospective, non-interventional Post-Authorization Safety Surveillance (PASS) program was initiated in 2004 to document ADVATE safety and efficacy in routine clinical practice. More than 900 subjects newly prescribed ADVATE have been enrolled in the US, EU and Japan alone. Herein, we present final US and interim EU results comprising 506 treated subjects, 451 of which have completed the 1-year observation period and 20 ongoing. Age cohorts were as follows: 27 neonates/infants (0-<2 years), 128 children (2-<12 years), 54 adolescents (12-<16 years), and 285 adults (≥16 years); 12 with age unreported. At study entry, 61 patients were considered previously untreated (PUP) or minimally treated (MTPs) defined as ≤50 previous exposure days [EDs]); 437 subjects were considered previously treated patients (PTPs) (>50 previous EDs). Eight subjects had unknown exposure history. Twenty-nine PUPs/MTPs reported exact exposure history prior to ADVATE: 5 reported 0 ED; 7 reported 1 to 3 EDs; and 17 reported 4 to 50 EDs. Most study subjects had FVIII ≤2%; 46 of which were PUPs/MTPs and 389 were PTPs. Among PTPs with FVIII ≤2%, 331 had no known history of inhibitor; 58 reported having a history of inhibitor prior to receiving ADVATE. At enrollment, 58% of subjects were prescribed prophylaxis, while 42% were treated on-demand. A total of 4848 bleeds in 343 subjects were treated. The on-demand efficacy was rated only as excellent/good in 94% of subjects. Subjects who were prescribed ADVATE prophylaxis (N=291) received a mean of 3 infusions per week at 29 IU/kg/ infusion. The median annual bleed rate on prophylaxis was 2.4 bleeds per year. Prophylactic efficacy assessments were provided for 268 subjects; 92% received only excellent/good ratings. Similar efficacy results were seen for the various populations with or without history of inhibitor. No unusual or unexpected AEs were seen in the study. One de novo, low-titer (1.4 BU), transient inhibitor was reported in 331 PTPs with FVIII ≤2% and no prior inhibitor history, providing a de novo inhibitor incidence estimate of 0.3% (95% CI, 0.01% – 1.67%). Two recurrent, low-titer inhibitors (≤1.5 BU) were reported in PTPs with FVIII ≤2%. One de novo, high-titer and 1 de novo, low-titer inhibitor were reported in PTPs with FVIII >2%. Two high-titer inhibitors were reported among 61 PUPs/MTPs. Other SAEs included 1 headache, 2 allergic reactions, 1 ineffective drug response in a subject with inhibitor and ITI history, and 1 hemarthrosis secondary to trauma. Results from the US and EU PASS registries validate the ADVATE safety and efficacy profile in the general hemophilia A population, in real-world clinical settings. Consistent with the formal clinical study findings ADVATE continues to demonstrate low risk for inhibitor development in PTPs.
A Prospective Evaluation of Bleeding Tendency and Efficacy of Antihemorrhagic Treatments in Patients with Increased Von Willebrand Factor (VWF) Clearance (Von Willebrand Disease Vicenza AND C1130F Mutation).
Authors: Castaman Giancarlo , Tosetto Alberto , Federici Augusto B. , Rodeghiero Francesco
Published: October 2019

Background. von Willebrand disease Vicenza (VWD-VI) is characterized by severely reduced FVIII and VWF levels, the presence of ultra-large multimers in plasma, heightened response to desmopressin and increased clearance of VWF after desmopressin. Increased clearance of VWF has been identified in other VWF mutations, such as C1130F with similarly low FVIII/VWF levels as in VWD-VI. So far, the bleeding risk of these patients has not been formally established and it is unclear whether they need a different treatment approach.

Aims, Design of the study, Patients and Methods. We prospectively evaluated the clinical history of 60 patients (32 M, 28 F) with VWD Vicenza (carrying R1205H mutation) and 23 patients (9 M, 14 F) with C1130F mutation, both characterized by increased VWF clearance. All in-hospital visits for bleeding symptoms, treatments and prophylaxis prior to invasive procedures were recorded together with home-treatments. All the remaining patients were contacted yearly to update their bleeding symptoms, if any. The study lasted from January 2002 to December 2007.

Results. During follow-up, 21/60 (35 %) and 9/23 (39 %) of patients with VWD-VI or C1130F respectively did not require treatment for bleeding or prior to invasive procedures. Ten patients with VWD-VI and 5 with C1130F had 1 to 3 tooth extraction instances treated with a single desmopressin infusion plus oral tranexamic acid for 5 days, without mishap. A single patient carrying out dental extraction without any prophylaxis had bleeding promptly stopped by a single desmopressin infusion. Four women with VWD-VI and 6 with C1130F had 1 or 2 pregnancies during follow-up, and successfully completed delivery by using two or three desmopressin infusions over 24-48 hours after vaginal delivery. Epistaxis requiring consultation or treatment was almost limited to pediatric age and a single adult patient only required treatment for an isolated episode. A single post-traumatic joint bleeding required a successful treatment with desmopressin. Only 3/15 (20 %) women in fertile age with VWD-VI required treatment for menorrhagia (iron suppletion, combined oral estroprogestinic pill, tranexamic acid) compared to 8/9 (89 %) with C1130F mutation. Most minor surgical interventions were successfully covered with desmopressin while major surgery required FVIII/VWF concentrates since prolonged hemostasis was required.

Conclusions. Spontaneous bleeding in patients with VWD-VI and C1130F appeared to be mainly limited to epistaxis in pediatric age and menorrhagia in females. Most of clinical situations are successfully manageable with desmopressin, as usually occurs for other type 1 VWD patients, and despite the short FVIII and VWF half-life post-infusion.
Effects of Age on the Development of Arthropathy in Mice with Hemophilia.
Authors: Valentino Leonard A. , Hakobyan Narine , Sumner D. Rick , Enockson Candace
Published: October 2019

The National Hemophilia Foundation, World Federation for Hemophilia and the World Health Organization recommend that prophylaxis be continued indefinitely. Questions about stopping prophylaxis as boys become adolescents or adults have been raised. In particular, discontinuing prophylaxis after a boy reaches skeletal maturity has been suggested. The impact of joint bleeding on these mature joints is thought to be less deleterious than in those of younger patients. To test the hypothesis that older, skeletally mature joints are less susceptible to joint damage after hemarthrosis, mice deficient in factor 8 gene expression (n=42) were studied to determine the impact of age and skeletal maturity on the development of hemophilic arthropathy. Mice at different ages were subjected to needle puncture of the knee joint capsule to induce a single massive hemarthrosis and then sacrificed 30 days or one year after the joint bleed. The diameter of the right (R) and left (L) knee joints was measured and the greatest bone diameter recorded. The joints were then isolated and subjected to image analysis using micro-computed tomography (mCT). Images were reviewed by 6 examiners blinded to the experimental conditions. After 30 days, a single episode of hemarthrosis resulted in a significant increase (p=0.0152) in the size of the affected joints compared to the control contralateral joint (figure). Although all ages of mice demonstrated bone hypertrophy, this was most pronounced in older mice. mCT images showed that changes were present in bones of the affected knees both young and old mice compared to the contralateral control joints. A pitted appearance of the surface was prominent in young mice early after a joint bleed (day 30). By one year, this pitting was much less evident. Osteocyte formation was a prominent feature. The surface contour of older mice scarified on day 30 was rugose. By one year, osteocyte and new bone formation dominated the surface. Consistent with the observation in patients with hemophilia, the patella of older mice was laterally displaced. The knee joints of both young, skeletally immature and older skeletally mature mice demonstrate marked structural changes after a single hemarthrosis. These results indicate:

that bone hypertrophy is greater in older mice; the blood-induced joint damage is different depending on the age of the animal; that bleeding into the skeletally mature joint damages the bones resulting in new bone formation; bleeding into immature joints causes more pitting of the surface and bone changes.

Therefore, in conclusion, this data does not support the hypothesis that bleeding into the mature joint is less detrimental compared to hemarthrosis in the younger skeletally immature joint. This observation, if translatable to humans suggests that joint bleeding in adults is likely to result in bone damage and that prevention of joint bleeding should be a goal for patients of all ages.

Figure Figure
A Single Point Mutation Gln716His on the α2 Integrin: The New Platelet Alloantigen Casa.
Authors: Bertrand Gerald , Jallu Vincent , Kervran Dominique , Martageix Corinne , Kaplan-Gouet Cecile
Published: October 2019

We report here the identification and characterization of a new low-frequency platelet alloantigen Casa involved in a case of neonatal alloimmune thrombocytopenia (NAIT). A 29-year-old mother gave birth to a full-term male infant who exhibited petechiae at birth. Nine hours post-delivery the platelet counts revealed a severe thrombocytopenia (16.109platelets/L) leading to platelet transfusion therapy associated with IVIG. The outcome was uneventful. Blood samples from the parents and infant were referred to our laboratory for investigation because of suspected NAIT. Maternal serum showed a specific positive reaction with the antigen-capture assay (MAIPA) only when it was tested with the paternal platelets and the monoclonal antibodies Gi9 (Immunotech, Marseille, France), P16 (NIBSC, Bristol, UK), and AK7 (Abcys, Paris, France) directed against the GPIa-IIa (a2b1 integrin). Nucleotide sequence analysis of GPIa cDNA of the father and newborn showed a nucleotide substitution at position 2235 (2235G>T according to the International Nomenclature). This substitution induces a Q716H amino acid change in the GPIa mature protein, located outside the I domain involved in cell-adhesion for collagen. In vitro analysis of recombinant CHO cells expressing wild-type or mutant (Q716H) human GPIa allowed us to demonstrate that this single amino-acid substitution is responsible and sufficient for inducing Casa antigen expression. Adhesion of CHO cells to collagen coated on microtiter plates was not modified by the Cas polymorphism, nor by the maternal anti Casa alloantibodies, indicating that the mutation does not affect the function of the integrin a2b1. PCR-SSP was developed for Casa genotyping. In a Caucasian population study none of the 100 unrelated blood donors was found to be Casa carrier. In conclusion, the Casa antigen described here, implicated in a case of severe neonatal thrombocytopenia is a low-frequency platelet antigen in the Caucasian population. This study highlights the high polymorphism of the GPIa gene.
Profiles of Th1, Th2, Th17 and Treg Cells in Patients with Chronic Idiopathic Thrombocytopenic Purpura.
Authors: Zhou Rong-Fu , Ou-yang Jian , Chang Da-Yu , Xu Jing-Yan , Chen Bing , Yang Yong-Gong , Zhang Qi-Guo , Shao Xiao-Yan
Published: October 2019

Objective: To explore the profiles of Th1,Th2, Th17 and Treg cells in patients with chronic idiopathic thrombocytopenic purpura.

Methods: Samples of peripheral blood were collected from 30 chronic ITP patients ( 9 males and 21 females), aged 41, 21 being in active stage, and 9 in remission stage, and 9 healthy persons in control (3 males and 6 females), aged 36. Peripheral blood was cultured, and activated with PMA/ionomycin when Th1, Th2 and Th17 cells were detected. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-γ, interleukin (IL)-4 and interleukin (IL)-17 so as to identify the Th1 cells (CD3+ CD8− IFN-γ+ IL-4− cells), Th2 cells (CD3+ CD8− IFN-γ − IL-4+ cells) and IL-17 cells (CD3+ CD8− IL-17+ cells); Treg cells were identified to CD4+ CD25+ Foxp3+ cells and uncultured peripheral blood was used to measured the CD4+ CD25+ Foxp3+ cells by flow cytometry. The ratios of Th1/Th2 were calculated.

Results: The Th1/Th2 ratio for patients in active stage was 15.04±9.67, significantly higher than those for patients in remission stage (7.17±5.38, P <0.05) and in control (8.47±3.78, P <0.05); the percentage of Treg cells of the patients in active stage was 0.89±0.58%, significantly decreased than those of patients in remission stage (6.41±1.86%, P <0.001) and in control (6.06±0.85%, P <0.001); the percentage of Th17 cells was 1.94±0.77% for patients in active stage, 2.16±0.52% for patients in remission stage and 1.82±0.58% for patients in control, respectively, and there was no statistic significance between them.

Conclusion: Chronic ITP is a Th1 predominant disease; decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in chronic ITP; Th17 cells might not play a role in the development of chronic ITP.
Maternal Immune Response to Fetal Platelet GPIbα Causes More Frequent Miscarriage in An Animal Model: A Potential Explanation for Low Reported Incidence of Fetal and Neonatal Immune Thrombocytopenia Mediated by Anti-GPIbα Antibodies.
Authors: Li Conglei , Piran Siavash , Chen Pingguo , Lang Sean , Ware Jerry , Ruggeri Zaverio M. , Freedman John , Ni Heyu
Published: October 2019

Fetal and neonatal immune thrombocytopenia (FNIT) is a life-threatening bleeding disorder, resulting from fetal platelet opsonization and destruction by maternal antibodies developed during pregnancy. The frequency of FNIT has been estimated at 0.5–1.5/1,000 liveborn neonates. However, the incidence of fetal mortality is currently unknown, as the rate of miscarriage in affected pregnant women has not been well studied. Integrin αIIbβ3 and Glycoprotein (GP) Ibα are major glycoproteins expressed on the platelet surface and are the two major antigens targeted by anti-platelet antibodies in autoimmune thrombocytopenia (ITP). However, it is unclear why the incidence of FNIT caused by anti-GPIbα antibodies is far lower than that of FNIT mediated by anti-β3 integrin antibodies. This difference cannot be well explained by the frequency of genetic polymorphisms of the two antigens. We hypothesized that: 1) GPIbα is less immunogenic, leading to less maternal antibody production during pregnancy, or 2) anti-GPIbα antibodies cause a less severe pathology, and thus have a lower chance of being reported, or 3) anti-GPIbα antibodies cause higher incidence of miscarriage, resulting in reduced reported cases. To test these hypotheses, the maternal immune response against fetal platelet GPIbα versus β3 integrin were compared in FNIT models, using syngeneic background BALB/c GPIbα−/− and β3−/− mice. The FNIT models were established by transfusing female GPIbβ −/− or α3−/− mice with 108 gel-filtered platelets from wild-type (WT) BALB/c mice weekly. After two platelet immunizations, flow cytometry assays were used to detect the titers of anti-GPIbα and anti-β3 antibodies, and the immunized females were bred with WT BALB/c male mice. We found that there was no significant difference in mean antibody titer between the two groups (P>0.05). However, miscarriage occurred more frequently in anti-GPIbα-mediated FNIT (14/16 versus 8/16, P<0.05), particularly in pregnant mice with antibody titer less than 1:800 (11/13 versus 6/14, P<0.05). When antibody titers were higher than 1:800, miscarriage occurred in all mice and no difference was observed between the two groups (3/3 versus 2/2, P>0.05). Our data suggest that fewer reported FNIT cases mediated by anti-GPIbα antibodies cannot simply be explained by less immunogenicity of GPIbα, or less severe pathology caused by anti-GPIbα antibodies. Higher incidence of miscarriage caused by maternal immune response to fetal GPIbα likely masks the reported frequency and severity of this life-threatening disease. The mechanisms leading to miscarriage in FNIT, and the potential therapeutic effect of intravenous immunoglobulin (IVIG) in this disorder are currently being investigated. (Li C and Piran S contributed equally to this work).
A Predictive Cost-Effectiveness Analysis of the Treatment of Chronic, Refractory Immune Thrombocytopenic Purpura (ITP) with Novel Agents: An Economic Model Based on Long-Term Treatment Data with Rituximab Versus Romiplostim.
Authors: Oatis Wendy , Shankaran Veena , Saghir Faisal , Bennett Charles L. , Richey Elizabeth , Bussel James B
Published: October 2019

Introduction : Novel non-surgical options for treatment of chronic immune thrombocytopenic purpura (ITP) are emerging. These include rituximab, an anti-CD20 monoclonal antibody, and romiplostim, a soon to be approved thrombopoeitic receptor agonist. However, long-term clinical data with these agents have not been reported previously and the cost of romiplostim treatment has not been announced. Herein, we report a “break-even” cost-effectiveness analysis, based on 2.5 years clinical outcome data for both agents, which yields a suggested market cost for romiplostim that would result in equivalent cost-effectiveness profiles for the two agents when administered as treatment for chronic ITP.

Methods: Effectiveness data were based on 2.5-years results from studies of chronic ITP patients treated with rituximab or romiplostim. We calculated the total costs of treating a cohort of chronic ITP patients with ritiuxan with splenectomy salvage (estimated cost of $8,100) as the therapeutic choice for treatment failures. We subsequently calculated a break-even price for romiplostin with splenectomy salvage that would result in a total cost of treating an ITP cohort equivalent to the costs of treating the rituximab/splenectomy cohort.

Results: At 2.5 years follow-up, a cohort of 100 chronic ITP patients initially treated with rituximab is associated with 40 therapeutic failures going on to splenectomy annually. Total treatment costs for this cohort is estimated at $2.2 million. At 2.5 years follow-up, a cohort of 100 chronic ITP patients initially treated with romiplostim is associated with 10 splenectomy failures per year. Total 2.5 year treatment costs for this cohort is $1.9 million for splenectomy plus the costs of treating 70 ITP patients with romiplastim for 2.5 years, 20 ITP patients for two years, and 10 ITP patients for one year. Hence, annual break-even costs for romipslatim are estimated at $20,000. The higher annual cost with romiplostim versus rituximab is related to better 2.5 year outcome rates. Hence, these cost estimates are preliminary, pending reporting of additional long-term studies.

Conclusions: It will be of interest to compare this value with cost-effectiveness estimates that are based on actual pricing of romiplostim (when the drug receives FDA approval). Policy makers would be wise to develop break-even cost estimates for novel drugs prior to FDA approval. These analyses may help inform initial price setting efforts.
Effect of Reducing Intravascular Hemolysis on Ferritin Homeostasis in Eculizumab Treated Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients.
Authors: Roeth Alexander , Schubert Joerg , Hock Christina , Christoph Sandra , Duehrsen Ulrich
Published: October 2019

BACKGROUND: In paroxysmal nocturnal hemoglobinuria (PNH), the lack of the GPI-anchored terminal complement inhibitor CD59 on erythrocytes renders these cells susceptible to continuous complement mediated hemolysis. Hemolysis leads to anemia, fatigue, red blood cell transfusions, renal impairment and increases the risk of thromboembolic events. Urinary iron loss is also common in patients with PNH due to intravascular hemolysis. Eculizumab is a terminal complement inhibitor that has been evaluated in two phase III studies. In previous studies eculizumab significantly reduced intravascular hemolysis (characterized by significant reduction of LDH levels) and significantly reduced transfusion requirements as well as thromboembolic events. In this study we evaluated the change in hemolytic parameters and levels of ferritin parameters in PNH patients treated with eculizumab in two centers (Essen and Homburg/Saar).

METHODS: Nineteen PNH patients were treated with eculizumab as follows: 4 x 600mg IV every 7±2 days; 900 mg 7±2 days later; and then 900 mg every 14±2 days. The median therapy duration was 31 months (range 1–40 months). Hemolysis, transfusion requirements and serum iron parameters were analyzed over time of treatment.

RESULTS: Eculizumab effectively inhibited intravascular hemolysis in all PNH patients as evidenced by an 79% decrease in LDH levels (mean±SD: 2,110±802 to 349±182 U/l (normal range: 100–247); p=0.0001) and reduced transfusion requirements. Persistent elevation of reticulocytes as well as the reduction of haptoglobin and hemopexin were observed in most patients, which suggest continuing extravascular hemolysis. Interestingly, we observed an 709% increase in ferritin levels in patients treated with eculizumab (median from 73 to 965 μg/l (normal range: 20–290 μg/L); p=0.001). This was more pronounced in patients still requiring some transfusions. Three patients were started on oral iron depletion therapy. No thromboembolic or serious adverse events were observed in eculizumab-treated patients. Two PNH patients were diagnosed with PNH-associated hematological disease (MDS, myelofibrosis).

SUMMARY/CONCLUSIONS: Eculizumab is safe and well tolerated in the analyzed cohort of PNH patients. Iron parameters in PNH patients treated with eculizumab should be monitored to determine if iron supplementation should be altered or iron depletion therapy should be considered. While some extravascular hemolysis may persist, intravascular hemolysis is effectively controlled with eculizumab and is associated with a concomitant improvement in anemia and quality of life.
Critical and Opposing Effects of T Cell-Derived TNFα and Interferon-γ on IL-17 Induction Assessed in Vitro and in Vivo Following Allogeneic Bone Marrow Transplantation
Authors: Li Minghui , Sun Kai , Hubbard Mark , Redelman Doug , Panoskaltsis-Mortari Angela , Nedospasov Sergei , Taub Dennis , Blazar Bruce , Welniak Lisbeth A. , Murphy William J.
Published: October 2019

IL-17-producing CD4 T cells (Th17) are a recently identified T helper subset that plays a role in mediating host defense to extracellular bacteria infections and is involved in the pathogenesis of many autoimmune diseases. In vitro induction of IL-17 in murine CD4+ T cells has been shown to be dependent on the presence of the proinflammatory cytokines TGF-β and IL-6 whereas IFNγ can suppress the development of Th17 cells. In the current study, we examined the roles of TNFα and IFNγ on IL-17 production by purified T cells in vitro and in vivo after allogeneic bone marrow transplantation (BMT). We present findings that expression of TNFα by the T cell itself is necessary for optimal development of Th17 under in vitro polarizing conditions. A novel role for T cell-derived TNFα in Th17 induction was observed when in vitro polarization of Tnf−/−CD4+ T cells resulted in marked reductions in IL-17+CD4+ T cells compared to Tnf+/+CD4+ T cells. In marked contrast, T cell-derived IFNγ markedly inhibited Th17 development as more IL-17+CD4+ T cells were found in Ifnγ−/−CD4+ T cells than in Ifnγ+/+CD4+ T cells, and of particular interest was the dramatic increase in IL-17+CD8+ cells from Ifnγ−/− mice. To determine if T cell-derived TNFα or IFNγ can regulate Th17 development in vivo we examined the differentiation of alloreactive donor T cells following allogeneic BMT. We have found that donor-derived Th17 cells can be found in lymphoid tissues and GVHD-affected organs after allogeneic BMT. However, transfer of Tnf−/− CD4+ T cells after allogeneic BMT resulted in marked reductions in Th17 cells in the spleen (18×103 vs 7×103, P<0.05). In agreement with the in vitro data and in contrast to what was observed with transfer of Tnf−/− CD4+ T cells, transfer of donor Ifnγ−/− T cells resulted in marked increases in not only IL-17+CD4+ but also IL-17+CD8+ T cells infiltrating the liver (7×103 vs 14×103, P<0.05; 4×104 vs 12.5×104, P<0.05). These results suggest that the donor T cell-derived TNFα and IFNγ opposingly regulate IL-17 induction of both CD4+ and CD8+ T cells in vitro and after allogeneic BMT which correlates with GVHD pathology.
Immune Mobilization with IL-2 and Growth Factors: Differential in Vivo Effects of the NKG2D Receptor on CD8+ and CD56+ Effector Cells
Authors: Meehan Kenneth R , Hill John M. , Ernstoff Marc , Sentman Charles L
Published: October 2019

Background: NKG2D, one of four NK cell activating receptors characteristically found on NK cells, has been identified on some CD8+T cells that mediate TCR-independent and non-MHC restricted tumor cell killing. These cytotoxic NKG2D+CD8+ effector cells aggressively kill myeloma cells. Since the NKG2D receptor recognizes ligands expressed on myeloma cells, NKG2D+ CD8+ effector cells provide a unique immunotherapy opportunity.

Methods: We designed an immune mobilization trial to mobilize autologous cytotoxic effector cells, with peripheral CD34+ hematopoietic progenitor cells. IL-2 (dose escalation) began on day 0 of mobilization and continued as a daily SQ injection for 11 days. On day 7 of mobilization, GM-CSF (7.5 mcg/kg/d) and G-CSF (5 mcg/kg/d) were initiated for 5 days (days 7–11). Leukapheresis was performed on Day 11.

Results: Eleven of 12 patients completed therapy (myeloma, n=11; NHL, n=1). One patient (NHL) progressed during treatment. The MTD of IL-2 was 6×105 IU/m2/d. All patients successfully mobilized and received an autologous transplant with normal engraftment (ANC recovery: day 13 median; range 10–14 days; platelet recovery: day 12 median; range of 0–13 days). When compared to baseline, the immune-mobilized cells demonstrate an increase in CD3+CD8+ T cells (p = 0.01), CD3+CD8+CD56+ T cells (p = 0.01) and CD56+ NK cells (p = 0.002). Cytotoxicity directed against a human myeloma cells increased from 9% (baseline) to 43% (p=0.02). Importantly, immune mobilization resulted in an IL-2 dose-dependent expression of NKG2D on CD8+T cells or CD56+ NK cells. Low dose IL-2 (6 × 105 IU/m2/d) moderately increased NKG2D expression on CD8+ T cells when compared to baseline (p = 0.02). High dose IL-2 (1.5 × 106 IU/m2/d) strongly enhanced NKG2D expression on CD 8+ T cells (p = 0.004). Low dose IL-2 (6 × 105 IU/m2/d) and high dose IL-2 (1.5 × 106 IU/m2/d) increased NKG2D expression on CD3-CD 56+ NK cells, with p values of 0.03 and 0.002, respectively. Preliminary data indicate these effects are sustained in vivo post-transplant.

Conclusions: Immune mobilization resulted in an IL-2 dose-dependent expression of NKG2D on CD8+T cells and CD56+ NK cells, thereby altering the cellular components of the mobilized cells. Since the NKG2D receptor recognizes ligands expressed on myeloma cells, NKG2D+ CD8+ effector cells provide a unique immunotherapy opportunity for the treatment of myeloma.