bluuebunny/crossref_metadata_2025 huggingface dataset:
“Age” of the Cord Blood (CB) Unit: Impact of Long-Term Cryopreservation and Storage on Transplant Outcome.
Authors: Scaradavou Andromachi , Stevens Cladd E. , Dobrila Ludy , Sung Dorothy , Rubinstein Pablo
Published: April 2020

Cryopreserved, banked CB is being increasingly used for allogeneic bone marrow reconstitution of unrelated transplant recipients. The effect of long-term storage on the viability and function of CB hematopoietic stem cells has been addressed only with in vitro assays and immunodeficient mouse models (Broxmeyer et al, PNAS2003; 100:645–50), but not in clinical transplantation. To evaluate the quality of CB units after prolonged storage in liquid nitrogen freezers, we compared the transplantation outcomes of patients that received CB grafts from the New York Blood Center NCBP stored for 8 years or more (Group 1, N=43, median time from unit collection to transplant: 9.2 years) to those transplanted with units stored < 2 years (Group 2, N=300, median time: 1.1 years). Patients were included in the study if they were transplanted in the period 2001–2006, received a single or double unexpanded unit graft and had no prior transplant in the 6 months preceding the CB graft. Double unit recipients were evaluated if the unit of interest was the one that engrafted, as demonstrated by post-transplant chimerism studies. The two groups were similar with respect to disease category (malignant diseases: 74% in Group 1, 69% in Group 2), location of transplant center (transplant in the US: 60% and 68%, respectively), use of non-myeloablative regimens (19% and 12%, respectively) and double unit transplants (14% and 10%, respectively). More patients in Group 1 were adults (42% and 27%, respectively, p = 0.035). Detailed information on CB thawing was available for all units in Group 1 and 217 in Group 2. Two units of the first Group and 3 of the second were reported to have problems at thawing (clots/leak). No immediate post-infusion complications were reported. Post-transplant ANC recovery was at a median of 25 days in Group 1 and 22 days in Group 2 (multivariate RR = 0.9, p = 0.6). Among patients who survived for 28 days or longer, 8/36 in Group 1 and 42/260 in Group 2 failed to achieve ANC500 (p = 0.3). Overall survival was not different in the two Groups (both 49% at 5 years). These results indicate that “old” CB units can be used effectively in clinical transplantation. Therefore, long-term cryopreservation of CB stem cells is feasible without compromising the quality and engraftment ability of the CB units, supporting the creation of a National Cord Blood Inventory with high quality units. However, these findings need to be viewed with caution: cryopreservation and storage procedures, equipment and devices vary significantly among different Banks. Thus, the results of the NCBP may not apply to all Cord Blood Banks.
Final Results of Lower Dose Fludarabine (F) and Cyclophosphamide (C), and High Dose Rituximab (R), (FCR-Lite) for Patients with Untreated Chronic Lymphocytic Leukemia (CLL).
Authors: Tarhini Ahmad A. , Land S. , Pietragallo L. , Laman A. , Sulecki M. , Marks S. , Meisner D. , Schaefer P.M. , Foon Kenneth A.
Published: April 2020

Introduction Standard FCR therapy in untreated CLL patients (F-25 mg/m2 d1–3 q 4wk; C-250 mg/m2 d 1–3 q 4wk; R-500 mg/m2 d1 q 4wk for 6 cycles) was reported to have complete remissions (CR) of 70% and overall responses (OR) of 95% (J Clin Oncol2005;23:4079). The major toxicity was grade 3/4 neutropenia during 52% of treatment courses. One approach to decrease neutropenia without compromising efficacy could be by reducing the doses of F and C and increasing the dose of R. Methods We conducted a phase II study for previously untreated advanced CLL patients treated with FCR-Lite (F-20mg/m2 d1–3 q 4 wk; C-150 mg/m2 d1–3 q 4 wk; R-500mg/m2 d1 and d14 q 4wks; maintenance R-500 mg/m2 ×1 q 3 months until progression). A Simon two-stage design was used where 15 patients were accrued in the first stage and because of acceptable toxicity and response rate in stage I an additional 35 patients were treated. The primary endpoint was response rate. Results A total of 50 patients were entered into this study and 42 are currently evaluable. There were 29 male and 13 female patients with an age range of 36–85 years (median 58) treated with a total of 236 courses of FCR-Lite. All 42 patients were evaluable for toxicity. Grade 3/4 neutropenia occurred during 29 (12%) courses with two episodes of neutropenic fever. One patient had cellulitis, another had pneumonia (not neutropenic). Grade 3/4 thrombocytopenia occurred during 7 (3%) courses and grade III/IV anemia during 6 (2.5%) courses. Among the 40 evaluable patients for response, the CR rate was 85%, PR rate was 15% with an OR rate of 100%. All of the CR patients were tested by flow cytometry and had <1% CD5+/CD19+ cells in their bone marrow after therapy. One patient with potential CR was excluded due to the absence of follow up bone marrow biopsy. Minimal residual disease (MRD) was tested by four color flow cytometry (sensitivity 0.01%) in 8 patients with CR (Genzyme Genetics Corp.). Seven had no evidence of MRD at 7, 8, 8, 14, 22, 25 and 30 months respectively, post CR, and one patient had 0.03% and 0.06% when tested at 12 and 18 months post CR respectively. Conclusions Our results in 42 patients suggest FCR-Lite is highly effective with considerably less grade 3/4 neutropenia than standard FCR. Complete responders had no detectable CD5+/CD19+ cells in their bone marrow following FCR-Lite. MRD testing is currently underway for all patients.
The Receptor for Hyaluronic Acid Mediated Motility (RHAMM): Characterization as an Immunotherapeutical Target in Chronic Lymphocytic Leukemia (CLL) and First Results of RHAMM-Derived Peptide Vaccination Trial.
Authors: Giannopoulos Krzysztof , Buhler Andreas , Chen Jinfei , Krober Alexander , Kowal Malgorzata , Dmoszynska Anna , Rolinski Jacek , Greiner Jochen , Dohner Hartmut , Stilgenbauer Stephan , Schmitt Michael
Published: April 2020

Background and Aims: The increased expression of receptor of hyaluronic acid mediated motility (RHAMM) was noted in CLL. The defective expression of RHAMM-exon4 splice variant that correlated with chromosomal instability was found to bear negative prognostic value in patients with multiple myeloma. Therefore, here we characterized expression of RHAMM with its splice variants and correlated it with prognosis in CLL. Moreover, based on our previous results we initiated a RHAMM-derived R3 peptide vaccination study for patients with B-CLL in early stages of disease.

Methods: Messenger RNA expression of RHAMM/CD168 was assessed by quantitative RT-PCR in blood samples from 65 B-CLL patients, at the time of diagnosis. The splice variant expression was measured using fluorescence capillary electrophoresis. Three hundred mcg RHAMM R3 peptide (ILSLELMKL) emulsified with the incomplete Freund’s adjuvant (ISA-51) day 3, as well as GM-CSF days 1–5, was administrated four times subcutaneously at a biweekly interval.

Results: The real-time RT-PCR results revealed higher RHAMM/TBP expression rates in patients with unmutated IgVH status compared with mutated CLL cases (0.047 vs. 0.029, p=0.012). Both splice variants RHAMMFL as well as RHAMM-exon4 showed significantly higher expression in unmutated CLL cases. Increased expression of RHAMM/TBP was noted in advanced stages of disease. A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. CLL patients with a RHAMM/TBP ratio >0.028 showed a significantly shorter median treatment-free survival (TFS) (16 vs. 38 months, p=0.0019). In a bivariate analysis, patients with VH mutated status and lower amounts of RHAMM presented longer TFS compared to unmutated cases with a high RHAMM expression (110 vs. 8 months, respectively). An enhanced RHAMM expression was observed in bone marrow samples of 5 B-CLL patients compared with the results obtained in blood (RHAMM/TBP 0.102 vs 0.032). Similar tendency to higher RHAMM/TBP ratios was observed after CD40L stimulation (RHAMM/TBP 0.025 vs.0.113). In five patients who completed all four doses of R3 peptide vaccine no toxicity related to vaccine was observed. Reduction of leukemic cells was noted in 3 patients but no clinical responses meeting NCI criteria was observed. Immunological responses were observed in patients who achieved hematological improvement. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, and its expression levels are correlated with the clinical course of the disease. RHAMM expression might indicate the proliferative potential of leukemic cells. First results of peptide vaccination using RHAMM-derived peptide showed limited reduction of tumor burden.
The Frequency of Rheumatic Disease Autoantibodies in Patients with ADAMTS13-Deficient Thrombotic Thrombocytopenia Purpura (TTP).
Authors: Swisher Karen K. , Lewis Qurana F. , James Judith A. , Hovinga Johanna A. Kremer , Lämmle Bernhard , Terrell Deirdra R. , Vesely Sara K. , George James N.
Published: April 2020

Patients who have TTP associated with acquired, severe ADAMTS13 deficiency have an autoimmune etiology and therefore may have increased risk for additional autoimmune disorders. The Oklahoma TTP-HUS Registry enrolled 247 consecutive patients with their first episode of clinically diagnosed TTP from 11-13-1995 (the date of our initial ADAMTS13 measurement) to 6-30-2006 for whom plasma exchange treatment was requested; ADAMTS13 activity was measured in 228 (92%) of patients immediately before their first plasma exchange treatment; 42 (18%) patients had ADAMTS13 activity <10%. Three patients were excluded from this analysis because of preexisting systemic rheumatic disease (systemic lupus erythematosus (SLE), 2, Sjogren’s syndrome, 1). To examine the potential risk for development of autoimmune disorders, we measured screening autoantibodies (ANA, dsDNA, Sm, nRNP, Ro, La, ribosomal P, Jo-1, anti-phospholipid (aPL) IgG and IgM) in 34 of the 39 (87%) remaining patients. The median age at initial presentation was 39 years (range 9–71 years); 27 (79%) patients were women; 13 (41%) patients were black. Autoantibodies were measured by indirect immunofluorescence, immunodiffusion, or ELISA. Measurements were performed only once in 16 patients; in 18 patients 2–3 measures were performed over a period of 13 to 126 months.

Rheumatic disease autoantibodies TTP patients *1 patient had a maximum titer of >1:3240 in 2 samples; 1 patient developed overt SLE and his titer decreased with treatment. ANA ≥1:40 on at least one occasion 33/34 (97%) ANA ≥1:120 on at least one occasion 29/34 (85%) ANA measured ≥2 times, increasing titer 14/16* (88%) Anti-dsDNA ≥1:30 12/34 (35%) Anti-Ro positive 14/29 (48%) Anti-Sm positive 1/34 (3%) Anti-nRNP positive 1/34 (3%) aPL IgG and/or IgM ≥moderate positive 4/34 (12%)

No patients had positive tests for anti-La, anti-Ribo-P, or anti-Jo-1. 23 (68%) of the 34 patients had a positive test for one or more rheumatic disease autoantibodies (dsDNA, nRNP, Ro, La, or moderately positive aPLs). 4 of the 34 patients died during their initial episode; the remaining 30 patients have been followed for a median of 6.4 years (range, 1–11.5 years). During this time only 1 patient has developed clinically evident SLE; no other patients have developed systemic rheumatic diseases.

Conclusions:

A high prevalence of rheumatic disease-associated autoantibodies were found in a cohort of consecutive patients with TTP associated with acquired severe ADAMTS13 deficiency. The presence of dsDNA and Ro autoantibodies and increasing ANA titers suggest that patients with ADAMTS13-deficientTTP may have a potential risk for developing additional autoimmune disorders such as SLE or Sjogren’s syndromes. [3] These serologic observations support clinical observations that presenting features of TTP and SLE may overlap.
Isolation and Characterization of CD4+ T Cells Specific for the HPA 1a Epitope Associated with Neonatal Alloimmune Thrombocytopenia.
Authors: Ahlen Maria T. , Killie Mette K. , Skogen Bjorn , Husebekk Anne , Stuge Tor B.
Published: April 2020

Neonatal alloimmune thrombocytopenia (NAIT) can cause severe complications such as intrauterine death or intracranial hemorrhage (ICH) in the newborn, and is caused by the transfer of platelet-depleting antibodies from the mother to the fetus during pregnancy. These antibodies react with allogeneic epitopes, most commonly human platelet antigen (HPA) 1a, when present on fetal platelets. Although these responses are thought to be a result of a T cell-dependent immune response, HPA 1a specific T cells have not yet been isolated. To examine whether HPA 1a specific T cells could be detected and isolated, we collected PBMC post delivery from an HPA 1a negative mother who gave birth to an HPA 1a positive neonate suffering from severe thrombocytopenia (platelet count <50×109/L). The cells were stimulated with HPA 1a peptides (20aa) in long term cultures supplemented with IL-7 and IL-2, and subsequently, IL-15. After 4 weeks in culture these cells were labeled with CFSE dye and restimulated with HPA 1a or control peptides. After additional 2 weeks in culture supplemented with IL-2 and IL-15, specific proliferative responses were detectable by CFSE dye dilution by flow cytometry. The cells were cloned by fluorescent-activated cell sorting (FACS) and expanded in numbers with anti-CD3 stimulation in the presence of irradiated allogeneic PBMC and IL-2. The resulting clonal T cell lines were characterized in proliferation assays, ELISPOT assays and phenotyped by flow cytometry. All clones were CD3+, CD4+ and CD19−, and the majority of the clones proliferated and secreted cytokines in response to stimulation with HPA 1a peptides, but not control peptides. In ELISPOT assays, peptide-pulsed antigen-presenting cells were required for T cell detection. These clonal HPA 1a specific CD4+ T cell lines represent formal evidence of the existence of HPA 1a specific T cell responses related to NAIT and will serve as important tools for further characterization of maternal immune responses associated with NAIT.
Serial Analysis of Hematopoietic Progenitors in Mpl−/− Mice.
Authors: Kaffanke Janet , Germeshausen Manuela , Welte Karl , Ballmaier Matthias
Published: April 2020

Thrombopoietin (TPO) is the pivotal regulator of megakaryocytopoiesis but also an important factor for early multipotent hematopoietic progenitors. Loss-of-function mutations in the human MPL gene coding for the TPO receptor are the cause of congenital amegakaryocytic thrombocytopenia (CAMT), a rare inherited bone marrow failure syndrome presenting as isolated thrombocytopenia at birth and progressing to aplastic pancytopenia during the first years of life. In contrast, Mpl−/− mice although exhibiting thrombocytopenia and severely reduced numbers of early hematopoietic progenitors do not develop any signs of pancytopenia or generalized bone marrow failure during lifetime. To identify parallels and differences in the impact of TPO on hematopoietic development between mice and men we serially analyzed subpopulations of hematopoietic cells from early progenitors to mature peripheral blood cells from Mpl−/− mice (kindly provided by Warren Alexander, WEHI, Melbourne, Australia) over a period of 24 months. We determined frequency, clonogenicity and proliferative potential of hematopoietic progenitors from different hematopoietic tissues. Compared to wild type mice we found a strong reduction of multipotent stem cells (HSC), of common myeloid progenitors (CMP), of megakaryocyte/erythrocyte lineage restricted progenitors (MEP) but also of common lymphoid progenitors (CLP) in Mpl−/− mice. We did not find any significant changes in the frequency of these cells over a period of 24 months. For the CMP and MEP subpopulations of myeloid progenitors we found not only a decreased frequency but also a reduced clonogenicity and proliferative capacity. Interestingly, these functional restrictions significantly increased significantly during aging, a finding that was similar to our results obtained with myeloid progenitors from CAMT patients. Impaired proliferation of myeloid progenitors rather than a reduced number could be the reason for development of pancytopenia in CAMT patients.
Rab38 Expression in Platelet Dense Granule Storage Pool Disease.
Authors: Ninkovic Ivana , White James G. , Stephens Kenyatta W. , Rangel-Fihlo Artur , Wu Francsisca C. , Datta Yvonne H.
Published: April 2020

Platelet dense granule storage pool disease (SPD) is a bleeding disorder characterized by a lack of normal platelet dense granule function, as evidenced by decreased platelet aggregation in response to ADP, epinephrine and collagen. Platelet SPD has been studied most extensively in humans and rodents with Hermansky-Pudlak syndrome (HPS), whose phenotype is a result of defects in granule trafficking, leading to oculocutanous albinism, lysosomal storage diseases, and platelet dysfunction. We have been characterizing the fawn-hooded hypertensive (FHH) rat, which has been previously shown to have a bleeding disorder consistent with a platelet SPD and some of the features of HPS. While the platelets in the FHH rat have normal alpha granules and lysosomes, they lack dense granules as assessed by transmission electron microscopy. Platelet flow cytometric analysis of GPIb and GPIIb indicated that the FHH platelets have normal surface expression of these adhesion proteins. The FHH rat has a mutation in the Rab38 gene at the ATG start site, which is associated with the bleeding disorder. Rab38 is part of a large family of GTPases, which are involved in granule formation and secretion. Western blotting of FHH tissues revealed that there is no expression of Rab38 protein. We have used confocal immunomicroscopy to assess Rab38 in platelet formation and function. In normal rat and human platelets, there was punctate expression of Rab38. There was no Rab38 staining detected in FHH platelets. In human megakaryocytic cell lines, Dami and HEL cells, there was punctate staining of Rab38 that was mainly in the periphery of the cells, with a variable amount of perinuclear staining. There was partial colocalization of Rab38 with serotonin and VWF, and with Lamp-3, a marker of lysosomes. The degree of colocalization varied between cells. There was no clear association of Rab38 with actin and tubulin in megakaryocytes. We also examined a cohort of patients with SPD, but not HPS, for mutations in Rab38. The entire coding region and intron-exon boundaries of the Rab38 gene were sequenced in 18 patient samples collected at Emory University for the CDC Women with Bleeding Disorders and Menorrhagia Study. Ten of the patients had platelet function defects documented by standard platelet aggregation studies, and eight had no identifiable platelet function defect. No mutations in Rab38 were detected. Whereas numerous known polymorphisms were identified and confirmed, there was no association of any of them with platelet function abnormalities. In conclusion, Rab38 is expressed in platelets and megakaryocytes and may interact with other granule proteins during megakaryocyte development. Failure to express Rab38 is associated with platelet dysfunction. Further studies are needed to determine its function in megakaryocytes and platelets, and to determine whether defects in Rab38 are a cause of platelet SPD in humans.
Classifying Leukemias Based on Epigenetic Alterations.
Authors: Jelinek Jaroslav , Estecio Marcos R.H. , Kondo Kimie , He Rong , Zavadil Jiri , Issa Jean-Pierre J.
Published: April 2020

Acute leukemia is caused by alterations of blood-forming stem cells leading to uncontrolled growth and diminished capacity to differentiate into mature functional blood elements. Beside genetic changes, epigenetic alterations are increasingly recognized as important events in the pathogenesis of leukemia. Cytosine methylation in CpG islands at gene transcription start regions can cause heritable gene silencing and have the same functional effects as inactivating mutations. Hundreds of genes may become epigenetically silenced in leukemia. While many of the methylated genes are not expressed in blood cells, silencing of genes critically important for control of stem cell self-renewal, proliferation, differentiation, and/or survival can contribute to the malignant phenotype. We used a genome-wide method to identify methylated genes by hybridizing a CpG island microarray with amplicons obtained by the methylated CpG island amplification technique (MCAM). We analyzed 10 leukemia cell lines with different cellular origin (myeloid cell lines KG1, KG1a, HEL, K562, and TF1; T lymphoid cell lines CEM and JTAg; and B lymphoid cell lines ALL1, BJAB, and Raji). On average, 266 genomic loci were found to be hypermethylated in these cell lines, ranging from 56 (KG1) to 483 loci (Raji), reinforcing the idea of extensive epigenome alteration in leukemia. Unsupervised hierarchical clustering showed distinct methylation pattern in the cell lines of lymphoid origin versus myeloid leukemia cell lines and a GM-CSF-dependent erythroleukemia cell line TF-1, justifying the use of methylation markers for uncovering of tumor-specific pathways of gene inactivation. There was a striking difference in the number of hypermethylated genes between two closely related myeloid leukemia cell lines: KG1 (56 methylated loci) and its undifferentiated variant KG1a (225 methylated loci). cDNA microarray analysis showed that deoxy-azacitidine treatment induced expression of genes differentially methylated in KG1a (DKKL1, GBX, HIVEP3, KCNAB1, KIAA1102, NAV2, NEIL1, and RAX) but not in KG1 cells where these genes were unmethylated. Finally, we used bisulfite PCR followed by pyrosequencing analysis to quantitatively measure DNA methylation of several genes detected by MCAM. Ongoing analyses of bone marrow samples from leukemia patients showed hypermethylation of the following genes: GDNF (in 4/22 [18%] AML and 7/20 [35%] ALL patients), HAND2 (in 5/22 [23%] AML and 7/20 [35%] ALL patients), HIVEP3 (in 9/22 [41%] AML and 6/20 [30%] ALL patients), MPDZ (in 2/6 [33%] AML and 15/20 [75%] ALL patients), and NEIL1 (in 2/20 [10%] AML and 1/12 [8%] ALL patients). Mapping of DNA methylation abnormalities may detect epigenetic markers important for leukemia classification and prognosis. Identification of pathways frequently silenced by DNA methylation may also suggest new targets for specific therapy.
Defining the Leukemia Epigenome: Distinct Genome Wide Histone H3 Modification Patterns Exist in AML, ALL and Healthy Hematopoietic Progenitor Cells.
Authors: Muller-Tidow Carsten , Homme Claudia , Klein Hans-Ulrich , Hascher Antje , Koschmieder Steffen , Becker Anke , Wang Yipeng , McClelland Michael , Stadt Udo zur , Battke Florian , Nieselt Kay , Thiede Christian , Ehninger Gerhard , Berdel Wolfgang E. , Dugas Martin , Serve Hubert
Published: April 2020

Aberrant transcriptional regulation plays a crucial role in the pathogenesis of acute leukemias. Chromosomal translocations and other mutations frequently affect transcription and chromatin regulation-associated genes in these diseases. Based on these findings we hypothesized that distinct global chromatin modification patterns exist that can distinguish between progenitor cells (CD34+ HSC) and acute leukemia as well as its subtypes. We used high density oligonucleotide ChIP-Chip assays querying more than 31,000 genomic loci to analyze global Histone H3 acetylation (H3Ac) and Lysine 9 (H3K9me3) trimethylation patterns each in a large number of AML (n=115), ALL (n=30), CD34+ HSC (n= 21) and peripheral blood cell (N=18) specimens. Class comparisons and predictions as well as unsupervised analyses were performed. Bioinformatic analyses led to histone modification maps across the genome with Histone H3 acetylation levels peaking around the predicted transcriptional start sites. More than 1000 loci differed in H3 acetylation and H3K9me3 between AML and CD34+ specimens (5% FDR). Among the regulatory classes over-represented among the altered genes were those involved in oncogenesis and cellular proliferation/differentiation. Histone H3 acetylation and H3K9me3 patterns also differed at hundreds of loci between ALL and AML samples (5% FDR). Genes involved in transcriptional regulation were significantly altered between the two leukemia subtypes. Specific differences in global chromatin modifications allowed support vector machine-based classification of CD34+ progenitor cells, AML, and ALL, with about 90% sensitivity and specificity based on Histone H3 acetylation patterns. A similar classification power was observed for H3K9me3. Within the AML patients, several groups of patients were identified that clustered together in unsupervised hierarchical cluster analysis due to similar histone modification patterns. These patterns did not primarily depend on patients′ karyotypes,, indicating that it may be possible to define some, as yet unknown, different types of AML based on chromatin modification. On a global scale, acute leukemias are associated with specific histone modification patterns that distinguish AML from ALL and CD34+ hematopoietic progenitors. Taken together, these first data on the leukemia epigenome provide the basis for improved understanding of genes involved in leukemia pathogenesis.
A New Plate Based Plasma von Willebrand Factor (VWF): Ristocetin Cofactor Activity Assay.
Authors: Chen Dong , Pruthi Rijiv K. , Nichols William L. , Heit John A. , Owen Whyte G.
Published: April 2020

Introduction: Assays of plasma von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) are essential for the laboratory diagnosis of von Willebrand disease (VWD). However, the current manual platelet aggregation/aggregometry method has relatively poor operating characteristics. Our goal was to develop a simple, accurate and sensitive platelet based assay.

Methods: VWF protein from normal or patient plasma was first captured on the polyclonal anti-VWF antibody coated plastic plate surface. VWF:RCo activity was assayed by the binding of mitotracker (a red fluorochrome)-labeled, formalin-fixed normal platelets in the presence of ristocetin (1mg/ml) and under agitation (200 rpm by vortex). The fluorescence from the bound platelets, measured by a fluorescence microtiter plate reader (Fluor4000, BioRad), was related to the calibrator plasma signal (6∼150% or IU/dL), and reported as % or IU/dL. We tested plasma samples from normal donors (n=16) and known VWD patients (type 1, n=6; type 2, n=9) based on clinical history and levels of plasma VWF antigen (VWF:Ag), VWF:RCo activity (manual platelet aggregation/aggregometry method), factor VIII activity and VWF multimer analysis.

Results: For both normal donors and VWD patients, VWF:RCo activities by the plate assay vs. manual platelet aggregation/aggregometry method correlated closely (R2=0.84), and VWF:RCo/VWF:Ag ratios did not differ significantly. Platelet binding to VWF-coated plastic wells is both VWF specific and ristocetin dependent.

Conclusions: Although this new platelet based VWF:RCo plate assay is still being optimized and validated, the preliminary data suggest that it is simple, accurate and sensitive.
Establishment of an International Registry of Patients with Inherited FXIII Deficiency.
Authors: Ivaskevicius Vytautas , Seitz Rainer , Kohler Hans P. , Muszbek Laszlo , Ariens Robert A.S. , Seifried Erhard , Ichinose Akitada , Nugent Diane J. , Oldenburg Johannes
Published: April 2020

Inherited factor XIII (FXIII) deficiency is a rare autosomal recessive disorder affecting approximately one out of one to three million people. FXIII deficiency is characterized by a lifelong bleeding tendency, impaired wound healing and spontaneous abortions in females. In 1993, the European Thrombosis Research Organization (ETRO) Working Party on FXIII initiated a Europe-wide questionnaire on inherited FXIII deficiency. Since 2005, the registry has been endorsed by the Factor XIII Subcommittee of the Scientific and Standardization committee (SSC) of the ISTH. The analysis of 104 European patients demonstrated that the most common bleeding symptoms were subcutaneous bleeding (57%) followed by delayed umbilical cord bleeding (56%), muscle hematoma (49%), hemorrhage after surgery (40%), hemarthrosis (36%), and intracerebral bleeding (34%). Prophylactic treatment was initiated in about 70% of all patients. FXIII-B subunit-deficient patients had a milder phenotype than patients with FXIII-A subunit deficiency.The most frequent mutation affecting the F13A gene was a splice site mutation in intron 5 (IVS5-1G>A).This mutation was found in eight (17%) of 46 analyzed families.The haplotype analysis of patients carrying the IVS5-1A allele was consistent with a founder effect. Recently, we created a new FXIII database website (http://www.f13-database.de) with information about FXIII proteins, genes, mutations and polymorphisms. This website also includes a new questionnaire. Information provided by this questionnaire will allow better understanding of the differences of diagnostic and treatment possibilities in various parts of the world, and it will help to understand the impact of reduced FXIII activity in heterozygous relatives and finally, it will generally increase our knowledge on this rare disease. We hope that our initiative to establish a new international FXIII registry will be actively supported by the community involved in caring for FXIII deficient patients.
FDG-PET in the Serial Assessment of Patients with Lymphoma in Complete Remission.
Authors: Zinzani Pier Luigi , Stefoni Vittorio , Ambrosini Valentina , Derenzini Enrico , Musuraca Gerardo , Marchi Enrica , Alinari Lapo , Fina Mariapaola , Tani Monica , Pellegrini Cinzia , Fanti Stefano , Baccarani Michele
Published: April 2020

FDG-PET role in the assessment of lymphoma patients is well established but only few papers evaluated the usefulness of FDG-PET during follow up.

Aim: to prospectively investigate the value of serial FDG-PET scans in the follow up of lymphoma patients in complete remission. All lymphoma patients who achieved a complete remission were prospectively enrolled in the study and scheduled for serial FDG-PET scans at 6, 12, 18 and 24 months; further scans were then carried out on annual basis (overall 421 pts, 160 pts with Hodgkin’s Disease (HD) and 261 pts with non-Hodgkin Lymphoma (NHL) were studied). All patients had a final assessment using other imaging procedures and/or biopsy and/or clinical evolution. FDG-PET findings were reported as positive, indeterminate or negative for relapse; after comparison with all available data, PET results were categorized as true positive (TP), true negative (TN), false positive (FP), indeterminate turned out to be relapse (I+) and indeterminate turned out to be complete remission (I-).

Results: PET documented relapse in 42 cases at 6 mo (14 HD (8.8%) and 28 NHL (10.7%); in 31 cases at 12 mo (14 HD (9.5%) and 17 NHL (7.3%); in 27 cases at 18 mo (6 HD (4.5%) and 21 NHL (3.2%); in 9 cases at 24 mo (3 HD (2.4%) and 6 NHL (3.2%); and in 5 cases at > 36 mo (2 HD (2.8%) and 3 NHL (6.5%). Out of 125 scans reported as positive for relapse, 109 turned out to be TP (PPV of 87%); no false negative scan was recorded, and in the great majority of cases PET detected the presence of relapse before clinical evidence. Our results confirm that FDG-PET is a valid tool for lymphoma patients follow-up. The higher incidence of relapse occurred in both HD and NHL quite early after complete remission (at 6 and 12 months for HD and at 6, 12 and 18 months for NHD), thus confirming the usefulness of performing FDG-PET scans at these times in order to identify recurrence. The role of serial PET at later times (after 18 months for HD and 24 months for NHL) was found less relevant.
NK Cells Induce Alloreactive Donor T Cell Apoptosis and Decrease Proliferation in GVHD Induction.
Authors: Olson Janelle A. , Leveson-Gower Dennis B. , Beilhack Andreas , Negrin Robert S.
Published: April 2020

Natural Killer (NK) cells have the ability to suppress graft-versus-host disease (GVHD) while inducing a graft-versus-tumor response (GVT) during allogeneic bone marrow transplantation (BMT). Previous studies in allogeneic BMT models have shown NK cell trafficking to and proliferation in lymphoid organs and GVHD target organs, which are also sites of donor T cell trafficking. This study aims to investigate the impact of NK cells on alloreactive, GVHD-inducing donor T cells. Interleukin-2 activated allogeneic NK cells isolated from C57Bl6 (H–2b) or FVB (H–2q) animals were transplanted along with T cell-depleted bone marrow into lethally irradiated BALB/c (H–2d) mice, followed 2 days later by luciferase-expressing CD4+ and CD8+ conventional T cells from the same donor strain (NK+Tcon group). Control mice received lethal irradiation and T cell-depleted bone marrow on day 0, and luciferase-expressing T cells on day 2 after transplant (Tcon group). Bioluminescence imaging of NK+Tcon mice revealed a significantly lower T cell bioluminescent signal (p=0.03 for FVB into BALB/c on day 6) than from Tcon mice. CFSE proliferation analysis of alloreactive T cells on day 3 after transplant showed no significant change in the percent of donor T cells that have divided in the spleen, and only a slight decrease in the percent of T cells that have divided in the lymph nodes when NK cells are present. However, at this timepoint 82% of the proliferating cells have divided past the third generation, in contrast to 64% in the NK+Tcon mice. Donor T cells in both groups become equally activated in vivo, expressing similar levels of the early-activation marker CD69. T cells re-isolated from NK+Tcon animals at day 5 stained 2 to 10-fold higher for the TUNEL apoptosis marker than those from Tcon mice in the mesenteric and peripheral lymph nodes, respectively (p<0.0001). Additionally, decreased numbers of T cells were re-isolated from the peripheral lymph nodes in the NK+Tcon group as compared to the Tcon group. This increase in TUNEL staining was not seen when the transplanted NK cells were isolated from a perforin-deficient donor. This indicates that NK cells in lymph nodes use a perforin-dependent mechanism to increase apoptosis in proliferating, alloreactive donor T-cells, which are syngeneic to the transplanted NK cells. Donor T cells re-isolated from the lymph nodes of transplanted mice up-regulate the NKG2D ligand Rae1γ as compared to naïve T cells, as shown by FACS. This suggests that NK cells may cause direct lysis of alloreactive donor T cells in vivo during GVHD induction, mediated by the NK cell activating receptor NKG2D. This study provides crucial mechanistic information regarding the function of NK cells in suppressing GVHD.
Gab1 Is Involved in Erythropoietin Receptor-Mediated Survival Signal through Activation of the Erk Pathway.
Authors: Fukumoto Tetsuya , Kubota Yoshitsugu , Kitanaka Akira , Waki Fusako , Imataki Osamu , Ohnishi Hiroaki , Ishida Toshihiko , Tanaka Terukazu
Published: April 2020

Erythropoietin (EPO) is required for the survival, proliferation and differentiation of erythroid progenitor cells. The scaffolding adaptor protein Grb2-associated binder-1 (Gab1) is tyrosine phosphorylated upon stimulation of EPO in several cell lines and erythroid progenitor cells, and interacts with signaling molecules such as SHP2 phosphatase and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). However, biological functions of Gab1 in EPO receptor (EPOR)-mediated signaling has not yet been established. In this study, to explore the biological functions of Gab1 in vivo, Gab1-deficient F-36P human erythroleukemia cells were generated by means of transfection of the expression vector of siRNA against Gab1. WST-1 assay revealed that growth of Gab1-deficient F-36P cells was reduced to 61% and 77%, respectively, 5 days after incubation with lower concentrations of EPO (0.001 and 0.01 ng/ml), compared with that of mock-transfected F-36P (F-36P-mock) cells. In contrast, growth of Gab1-deficient F-36P cells at sufficient concentration of EPO (10 ng/ml) was similar to that of F-36P-mock cells. Analysis of apoptosis by flow cytometry using FITC-labeled annexin-V showed that the percentage of annexin-V-positive apoptotic cells in Gab1-deficient F-36P and F-36P-mock cells was increased to 19% and 34%, and 8% and 17%, respectively, 72 h after incubation with 0.01 and 0.001 ng/ml of EPO. These results indicate that Gab1 plays a crucial role in transducing EPOR-mediated survival signals. Next, we examined the molecular mechanism of EPOR-mediated signaling involved in survival of erythroid cells through Gab1. Western blot analysis showed that EPO-induced phosphorylation of threonine 202/ tyrosine 204 on Erk-1 and Erk-2 in Gab1-deficient F-36P but not in F-36P-mock cells was significantly suppressed. Interestingly, phosphorylation of serine 473 on Akt in Gab1-deficient F-36P cells in response to EPO was slightly suppressed in comparison with that in F-36P-mock cells. Therefore, Gab1-mediated survival signals appear to be mainly transmitted to downstream through activation of the Erk pathway, although the PI3K/Akt pathway may be involved in EPO-initiated survival signal transduction mediated by Gab1. Furthermore, EPO induced the association of SHP2 with EPOR in Gab1-deficient F-36P cells. Gab1 was associated with SHP2 in EPO-treated F-36P cells. In addition, Gab1 was constitutively associated with Grb2 in F-36P cells. Taken together, EPO induces the recruitment of Gab1 to EPOR through binding of Gab1 to SHP2, which is associated with EPOR. Because the guanine nucleotide exchange factor SOS1 is known to bind to the SH3 domain of Grb2, SOS1-Grb2 complex is recruited to vicinity of Ras at the plasma membrane to activate this GTP-binding protein through the interaction of Grb2 with Gab1, leading to activation of Erk.
Oral Treatment with PBI-1402 Increases Hemoglobin Level and Red Blood Cell Count: A Novel Approach to Treating Chemotherapy-Induced Anemia.
Authors: Gagnon Lyne , Barabe Jean , Penney Christopher , Roy Denis-Claude , Kovcin Vladimir , Bosnjak Snezana , Laurin Pierre
Published: April 2020

Background: PBI-1402 is a novel orally active low molecular weight synthetic compound with erythropoiesis stimulating activity. Furthermore, a clinical phase I study showed that PBI-1402 induced a significant increase (100%, p < 0.0001, compared to placebo) of relative and absolute reticulocyte count in healthy volunteers after 21 days of oral treatment and was devoid of significant side effects.

Outcomes: The objectives of this clinical phase Ib/II trial were to study the safety and tolerability of PBI-1402 and to assess its biological efficacy on hemoglobin (Hb) level and red blood cell (RBC) count in patients with Chemotherapy-Induced Anemia (CIA).

Methods: An open label phase Ib/II trial, monitored by a US CRO (Pharm-Olam International), was conducted in patients developing anemia after chemotherapy treatment. Three cohorts of 6 CIA patients received 8 weeks of treatment with PBI-1402 once a day, at different doses, and were monitored every two weeks for safety, tolerability, Hb level, RBC count and blood chemistry. Patients remained on their chemotherapy during PBI-1402 treatment.

Results: To date, 12 CIA patients have completed their PBI-1402 treatment. 83% of patients demonstrated a significant increase in RBC (p = 0.015) and 66% in Hb (p = 0.038). Among the responders, the mean Hb increase was 1.1 g/dL (p = 0.0007) from a baseline Hb value of 9.8 g/dL. No patient required a blood transfusion and only one patient had an Hb content below 9 g/dL (8.9 g/dL). PBI-1402 was well tolerated and no significant side effects were observed.

Conclusion: PBI-1402, via a mechanism of action distinct from erythropoietin, induced sufficient erythropoiesis to raise the RBC level and Hb in CIA patients. In addition, PBI-1402 is safe and well tolerated. PBI-1402 offers the potential for a novel therapy of the anemia of CIA.
A Unified Mechanism of Tyrosine Kinase Regulation by the Gatekeeper Residue.
Authors: Azam Mohammad , Seeliger Markus , Gray Nathanael S. , Kuriyan John , Daley George Q.
Published: April 2020

Protein kinase inhibitors achieve binding specificity through contact with a hydrophobic pocket within the ATP-binding cleft. Access to the pocket is dictated by an amino acid residue dubbed the “gatekeeper”, which is threonine in many kinases. Drug resistance is often due to substitution of the gatekeeper threonine with a bulkier amino acid residue that sterically hinders the binding of some drugs. Here we show that substituting a bulky hydrophobic residue in place of threonine in the native cellular forms of c-ABL, c-SRC, PDGFRA, PDGFRB, and EGFR activates kinase activity and promotes malignant transformation of BaF3 cells. Structural modelling and crystallographic analysis reveal that a network of hydrophobic interactions characteristic of the active kinase conformation is stabilized by the gatekeeper substitution. In the inactive kinase conformation, this hydrophobic interactions are dismantled. A small molecule inhibitor that maximizes complementarity with the dismantled spine (Compound #14) inhibits the gatekeeper mutation of BCR/ABL (T315I). These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases, and provide structural insights to guide the development of novel inhibitors.
Expression of G-CSF Receptor during Embryonic Development in a Csf3r-Cre Knock-In Mouse Model.
Authors: Touw Ivo P. , Valkhof Marijke , Erkeland Stefan J. , Meijers Karel , Danen-van Oorschot Astrid
Published: April 2020

G-CSF is the major regulator of neutrophil development and controls the proliferation, differentiation and survival of myeloid progenitors in both steady state and “emergency” granulopoiesis. More recently however, novel activities of the G-CSF/G-CSF receptor (CSF3R) axis have been suggested, especially after severe forms of tissue damage. For instance, upon myocardial infarction, G-CSF was shown to protect cardiomyocytes from apoptosis, reducing the extent of myocardial damage. Likewise, in ischemic stroke models, G-CSF treatment reduced infarct volumes by promoting neuronal survival and differentiation of neural stem cells in the brain. Finally, G-CSF has been suggested to promote hepatic regeneration after severe liver damage by directly stimulating the proliferation of oval cells. To study whether and to what extent CSF3R expression in damaged tissues reflects reactivation of a normal development program, we generated a csf3r-Cre knock-in model. To this end, the csf3r locus was targeted with a construct containing the cre gene and a puromycin selection cassette flanked by a 2.7 kb fragment of the csf3r 5′UTR and a 4 kb fragment directly downstream of the initiation codon of csf3r. Correct insertion of the cre gene was confirmed by Southern blotting and sequence analysis and subsequent crossing with FLP deleter mice resulted in successful germ line transmission and deletion of the puromycin cassette. These mice were crossed with the R26R LacZ reporter mouse and β-galactosidase (β-gal) activity was examined by FACS and histochemical analysis. At E9.5, no β-gal activity was detected, indicating that csf3r is not expressed at that stage of development. In contrast, in E12.5 embryos, β-gal staining was observed in fetal liver, heart, kidney, brain and intestine. Analysis of adult tissues showed that cardiomyocytes were β-gal positive in a mosaic pattern. A similar mosaicism was observed in liver, kidney and the intestines. In liver both hepatocytes and vascular endothelial cells stained positive, whereas in the kidney β-gal staining was detected in vascular endothelium, glomeruli and afferent and efferent vessels. In ileum and colon, β-gal activity was mainly seen in the stem and progenitor cell-containing crypts and the developing progeny migrating upwards along the villus. In bone marrow, high levels (>40%) of β-gal activity were detected in Gr1+ and CD11b+ myeloid cells, CD19+ (B) and CD3ε+ (T) lymphoid cells, NK cells and CD11c+ dendritic cells, indicating that G-CSFR was expressed in a common progenitor of these cell types. In agreement with this, β-gal activity was also detected in the primitive LSK population. In conclusion, our data indicate that CSF3R is expressed in both hematopoietic and multiple non-hematopoietic organs from embryonic development stage E12.5 onward. This may have important ramifications for a more wide-spread application of G-CSF in tissue regeneration.
Cobblestone Area-Forming Cell (CAFC) Content of Cord Blood Does Not Predict Engraftment in Double Cord Blood Transplantation.
Authors: Robinson Simon N. , de Lima Marcos , Yang Hong , Decker William K. , Xing Dongxia , Steiner David , Ng Jingjing , Thomas Michael W. , Champlin Richard E. , Hosing Chitra M. , Simmons Paul J. , Brouard Nathalie , Kidd Shannon , Cooper Laurence , Zweidler-McKay Patrick A. , Shpall Elizabeth J.
Published: April 2020

INTRODUCTION: Delayed engraftment remains a serious problem following cord blood (CB) transplantation. It may be due, at least in part, to the limited dose of CB hematopoietic stem and progenitor cells (HSPC) transplanted. Limitations associated with HSPC dose may be reduced by the transplantation of 2 CB units. In an accompanying abstract, de Lima et al. report in detail on an ongoing MDACC randomized study in which patients received double CB units as either 2 unmanipulated units (2×UN), or 1 unmanipulated and 1 ex vivo expanded CB unit (UN+EX). This study has revealed that one CB unit ultimately predominates as the source of long-term, sustained hematopoiesis. We hypothesized that the number of primitive HSPC in the CB unit might ultimately predict which CB unit would ultimately prevail. The in vitro cobblestone area-forming cell (CAFC) assay was used to provide a measure of primitive components of the CB HSPC in each unit. A photomicrograph of a typical cobblestone area (CA) derived from a single CB-derived CAFC is shown. (Figure) CA persisting in in vitro culture for ≥6 weeks (derived from CAFCwk6) represent relatively primitive HSPC and their numbers may provide a qualitative profile for CB units. We hypothesized that of the two CB units transplanted, the one with the greater number of CAFCwk6 would ultimately persist long-term in the patient.

METHODS: Clinical samples of 2×UN or UN+EX CB cells were plated in the in vitro CAFC assay.1 The frequency of CAFCwk6 was estimated and total CAFCwk6 numbers transplanted for each CB unit calculated. CAFCwk6 data for each CB unit and engraftment data from patients were analyzed to determine whether the number of CAFCwk6 transplanted was predictive of which CB unit would ultimately be responsible for long-term, sustained engraftment.

RESULTS: Preliminary data was accrued from 10 patients.(Table) Six patients received 2×UN and 4 patients received UN+EX (EX indicated by #). CAFCwk6 content at transplant was predictive of which CB unit would ultimately be responsible for long-term, sustained engraftment in only 6 of the 10 cases (60%).

CONCLUSION: CAFCwk6 represent a relatively primitive component of the HSPC compartment, however, these data suggest that CAFCwk6 numbers, although possibly a qualitative measure, are not a predictor of long-term sustained engraftment.

Figure Figure