bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Post-Transplant Lymphoproliferative Disorder (PTLD) with Rituximab or Chemotherapy: The University of Pennsylvania Experience.
Authors: Elstrom Rebecca L. , Andreadis Charalambos , Aqui Nicole , Tsai Donald E.
Published: April 2020

Background: PTLD is a rare but life-threatening side effect of organ transplantation. While some patients respond to reduction in immunosuppression (RI), in many patients this approach is ineffective or impractical. Chemotherapy and anti-B cell monoclonal antibody therapy have been explored for treatment of such patients, but most reports include small numbers of patients. We report the results of treatment of patients with PTLD with rituximab and/or chemotherapy at the University of Pennsylvania Medical Center.

Methods: Data were collected using retrospective chart and database review. Patients were evaluated for baseline characteristics as well as response to therapy, time to treatment failure, and overall survival. Prognostic factors were identified by univariate analysis.

Results: 35 of 117 adult solid organ transplant patients diagnosed with PTLD that were refractory to RI underwent treatment with rituximab and/or chemotherapy. Median time to PTLD was 4 years (range 3 months to 23 years), and most patients presented with advanced stage (57%) and extranodal involvement (74%). 71% had elevated LDH. There were no significant baseline differences between patients receiving rituximab and those receiving chemotherapy. 22 patients underwent treatment with rituximab. The ORR was 68% (CR 59% and PR 9%). Median time to treatment failure (TTF) was not reached at median follow up of 19 months (range 0.8–51 months), and estimated OS was 31 months (1.5–51 months). EBV positivity was a favorable prognostic factor for achievement of response and TTF (p<0.01). LDH elevation predicted shorter overall survival (0.04). No patient died due to rituximab toxicity, and all 8 patients who either did not respond or relapsed were able to undergo further treatment with chemotherapy. 23 patients received chemotherapy, 22 of whom were evaluable. ORR was 74% (CR 57% and PR 17%). At a median follow up of 27 months, median TTF was 10.5 months (0.4–54 months), and estimated OS was 42 months (0.4–70 months). Adverse prognostic factors for response included advanced stage (p=0.02), elevated LDH (p=0.01) and allograft involvement by tumor (p<0.01). These factors, in addition to lack of achievement of CR, also predicted poor overall survival (p<0.05 for all factors). 26% of patients receiving chemotherapy died due to treatment-related toxicity.

Conclusion: Rituximab and chemotherapy are effective treatments in patients with PTLD who fail or do not tolerate RI. While rituximab is generally well tolerated, chemotherapy is associated with marked toxicity. When possible, PTLD patients requiring therapy beyond RI should be considered for rituximab, especially those with EBV-positive disease. Chemotherapy should be reserved for those patients who fail rituximab, have EBV-negative tumors, or need a rapid response.
Genetic and Phenotypically Heterogeneity of Patients with Congenital Neutropenia.
Authors: Zeidler Cornelia , Schwinzer Beate , Bolyard Audrey A. , Pracht Gusal , Alter Blanche P. , Bonilla Mary A. , Cham Bonnie , Fier Carol , Freedman Melvin H. , Kannourakis George , Kinsey Sally , Boxer Lawrence , Dale David C. , Welte Karl
Published: April 2020

Severe congenital neutropenia (CN) is a general term for a group of disorders characterized by extremely low blood neutrophil counts (ANC < 0.5 x 109 ), early stage maturation arrest of myelopoiesis, and recurrent bacterial infections. More than 90% of CN patients respond to daily G-CSF treatment with a sustained neutrophil increase associated with a significant reduction of infections and improved quality of life. However, prolonged survival unmasks an increased risk of leukemic transformation in some, but not all, subcategories of CN patients.

The Severe Chronic Neutropenia International Registry (SCNIR) has collected longitudinal data on on more than 400 patients with various causes of CN. This unique resource allows classification of different subtypes of CN and estimation of the relative frequency of these conditions. Our new classification scheme is as follows:

By inheritance - autosomal dominant severe congenital neutropenia (ADCN), autosomal recessive (ARCN, Kostmann syndrome), sporadic CN. By genetic aberrations - ELA2 related CN, SBDS related CN-(Shwachman-Diamond syndrome), G 4.5 on Xq28related neutropenia (Barth syndrome), CXCR4 related neutropenia (myelokathexis and WHIM syndrome). By clinical phenotype - associated symptoms (e. g. metabolic disorders, such as Shwachman-Diamond syndrome, glycogen storage disease 1b, Barth syndrome), G-CSF responsive or non-responsive CN, with or without G-CSF receptor mutations, with or without osteoporosis, or dysplastic features (e. g. organ abnormalities). By ethnic origin - e.g. CN in consanguineous Kurdish families, recessive neutropenia in Swedish family (Kostmann syndrome).

Recent research and the work of the SCNIR now permit a much improved classification of CN. The identification of subtypes of CN, their distinctive risks of malignant transformation, and their responses to treatment has contributed substantially to our general understanding of the problem of neutropenia. This knowledge also now allows clinicians to give patients and families much improved prognostic information and better guidelines for therapy.
Alloimmunisation of Patients Undergoing Bone Marrow Transplantation in the Setting of Leucodepleted Apheresis-Collected Single Donor Platelets.
Authors: Crawford Julie H. , Hughes Annette , Cannell Paul K. , Herrmann Richard
Published: April 2020

Introduction: Immune platelet transfusion refractoriness often leads to requirements for specialised platelet products. Our patients are screened weekly for the presence of platelet antibodies during bone marrow transplantation (BMT) protocols and are routinely issued leucodepleted (LD) packed red cells and apheresis-collected leucodepleted single donor platelets (LDSDP).

Aims: We assessed the alloimmunisation rate in BMT patients, the impact of this on transfusion requirements and addressed the value of continued routine platelet antibody (PAI) screening in the setting of LDSDP.

Results: Routine PAI screens performed on 148 BMT patients over 33-months (January 2002–September 2004) were reviewed; 71 autologous, 55 sibling allogeneic and 22 matched unrelated donor (MUD) BMT patients. Thirteen patients were alloimmunised (antibody specificity anti-HLA (11 patients) and anti-HPA/platelet glycoprotein (2 patients)). The overall alloimmunisation rates were 8.8% (sibling allogeneic 14.5%, MUD 9.1% and autologous 4.2%). Alloimmunisation rates for women were 4.15 times higher than for male patients. Risk factors for secondary alloimmunisation were present in 92.3%. Seven of 9 alloimmunised women had children, 1 of 2 nulliparous women and all 4 male patients had extensive transfusion histories (all 5 were also transfused at other centres where LDSDP were not standard). Alloimmunisation was detected earlier in females, usually on first PAI screen and before transfusion. Alloimmunised patients required specialised platelet products (crossmatched, HLA, directed donations or frozen-thawed SDP). These measures resulted in improved post platelet transfusion increments.

Conclusions: Alloimmunisation continues to occur despite use of leucodepleted transfusion products. Evidence supporting secondary alloimmunisation was present in the great majority of patients with more female patients alloimmunised largely due to their parity. Secondary alloimmunisation is not prevented by transfusion of leucodepleted blood products, however early detection of alloimmunisation assists transfusion decisions. There is indication from this data that platelet antibody could be limited to a PAI screen at presentation (together with HLA typing) in patients who have been transfused or pregnant with a repeat PAI performed following transfusion. Further testing could be delayed until platelet transfusion refractoriness is suspected.
Non-Genotoxic Targeting of p53-Dependent and p53-Independent Signaling Pathways Efficiently Kills Hodgkin/Reed-Sternberg Cells.
Authors: Janz Martin , Stühmer Thorsten , Dörken Bernd , Bargou Ralf
Published: April 2020

Although the majority of patients with classical Hodgkin lymphoma (cHL) can be cured by conventional chemotherapy, a substantial proportion of patients will finally develop treatment-induced secondary malignancies. Therefore, non-genotoxic targeting of major cellular survival pathways could be an interesting novel treatment strategy for patients with cHL. In this study, we focused on the analysis of p53-dependent and p53-independent signaling pathways in Hodgkin/Reed-Sternberg (HRS) cells. To test whether p53 signaling is functional in cHL and whether activation of the p53 pathway is sufficient to kill HRS cells, we employed a recently developed small-molecule antagonist of MDM2, designated nutlin-3a, that disrupts the p53-MDM2 interaction. Nutlin-3a efficiently increased the level of p53 and induced expression of p53 downstream targets in Hodgkin cell lines with wild-type p53, whereas no effects were observed in Hodgkin cell lines that harbor p53 mutations. Activation of the p53 pathway led to strong induction of apoptosis in p53 wild-type Hodgkin cell lines. Knock-down of p53 by RNA interference protected cells from nutlin-induced apoptosis, demonstrating that nutlin-3a exerts its effects strictly through p53. In addition, MDM2 inhibition strongly sensitized HRS cells to cytotoxic drugs, such as doxorubicin, etoposide, or vincristine. In view of the fact that HRS cells are characterized by high constitutive NF-κB activity, we also analyzed the effects of a second non-genotoxic agent, geldanamycin, which is an inhibitor of the HSP90/NF-κB pathway. Titration experiments showed that the pro-apoptotic effects of geldanamycin correlate with the mutation status of IκB proteins, demonstrating strong induction of apoptosis in cell lines with wild-type IκB. Furthermore, Hodgkin cell lines that contain wild-type IκB but lack functional p53 (through mutation or siRNA knock-down) are resistant to nutlin treatment, but still respond to treatment with geldanamycin. This indicates that inhibitors of HSP90 induce apoptosis in HRS cells in a p53-independent manner. Therefore, combined targeting of p53-dependent and p53-independent pathways could be a promising approach to develop highly effective and less genotoxic treatment strategies for patients with cHL.
BIC and miR-155 Are Highly Expressed in Hodgkin, Primary Mediastinal and Diffuse Large B Cell Lymphomas.
Authors: Kluiver Joost , Poppema Sibrand , de Jong Debora , Blokzijl Tjasso , Harms Geert , Jacobs Susan , Kroesen Bart-Jan , van den Berg Anke
Published: April 2020

In a previous study we demonstrated high expression of the non-coding BIC gene in the vast majority of Hodgkin’s lymphomas (HLs). It is now known that BIC is a primary microRNA (pri-miRNA) that can be processed to miRNA-155. We analyzed HL and NHL cell lines and tissue samples to determine miR-155 expression levels. High levels of miR-155 could be demonstrated in all HL tissue samples and most cell lines. Most NHL subtypes were negative for BIC as determined by RNA-ISH. However, 8/8 cases of primary mediastinal B cell lymphoma (PMBL) showed BIC staining in 12% of the tumor cells. QRT-PCR analysis confirmed high relative BIC levels (35). 12/18 cases of DLBCL showed a variable BIC staining with on average 7% of positive cells. Interestingly, activated B cell-like (ABC-like) DLBCL showed on average 12% of tumor cells positive for BIC while germinal centre B cell-like (GCB-like) DLBCL only showed on average 4% of cells positive for BIC. Differences in BIC expression levels were confirmed by qRT-PCR analysis revealing average BIC levels of 22 for ABC-like and 12 for GCB-like DLBCL. Northern blot analysis showed expression of miR-155 in all DLBCL and PMBL derived cell lines and tissue samples analyzed. In summary, we demonstrate expression of pri-miRNA BIC and its derivative miR-155 in HL, PMBL and DLBCL. In DLBCL, BIC expression is most pronounced in the ABC-like phenotype, possibly as a result of NF-κB activation.
Bone Marrow Mast Cells Are Significantly Increased in Patients with Waldenstrom’s Macroglobulinemia, and Their Number Following Therapeutic Intervention Is Dependent on Extent of Response.
Authors: Santos Daniel Ditzel , Chemaly Mariana Z.A. , Tournilhac Olivier , OConnor Kelly E. , Hatjiharissi Evdoxia , Leleu Xavier , Xu Lian , Branagan Andrew R. , Manning Robert J. , Patterson Christopher , Ho Allen W. , Hunter Zachary R. , You James M. , Tai Yu-Tzu , Kutok Jeffery L. , Anderson Kenneth C. , Munshi Nikhil , Treon Steven P.
Published: April 2020

INTRODUCTION: In previous studies, we demonstrated that mast cells (MCs) may support the growth and survival of lymphoplasmacytic cells (LPC) in WM patients through expression of several ligands including CD40L, B-LYS, PDGFa and VEGF. While increased numbers of MC, as well as their association with LPC in the bone marrow (BM) of patients with WM has previously been reported, their enumeration before and following therapeutic intervention remains to be clarified.

METHODOLOGY: We therefore enumerated MC using tryptase staining in whole sections of BM trephine biopsies before and after therapy for 19 patients (median age 59; range 49–85 years) with the consensus panel diagnosis of WM who received rituximab (n=4), rituximab plus fludarabine (n=14) or bortezomib (n=1). Biopsies from 7 non-WM, age matched patients with normal trilineage myelopoiesis and who demonstrated no evidence of disease were used as controls.

RESULTS: At baseline, an increased number of BMMC was observed for WM patients (median 49/mm2; range 0.4–149.5/mm2) versus control patients (median 14/mm2, range 3.8–31.4/mm2); p=0.003. Among non-responding patients, the median number of BMMC rose from 42.76/mm2 (range 23.3–134.6/mm2) to 72.59/mm2 (range 59.1–77.9/mm2, n=4), while it remained stable for minor responders 14.9/mm2 (range 0.4–26.6/mm2) to 15.8/mm2 (range 11.9–44.8/mm2, n=3), and decreased for major responders (CR and PR) from 56.6/mm2 (range 7.1–149.5/mm2) to 34.13/mm2 (range 3.3–110.9/mm2, n=12).

CONCLUSION: These preliminary studies demonstrate that BMMC are significantly increased in patients with WM, and their number following therapeutic intervention is dependent on extent of response.
No Association between Chlamydia Psittaci and Ocular Adnexal Lymphoma in North American Samples.
Authors: Zhang Grace S. , Winter Jane N. , Variakojis Daina , Reich Steven , Lissner Gary S. , Bryar Paul , Regner Mary Ann , Mangold Kathy , Kaul Karen
Published: April 2020

Previous studies linked infection by Chlamydia psittaci to ocular adnexal lymphomas in Italian patients (Ferreri et al., JNCI 2004). Patients treated with doxycycline to eradicate the organism have demonstrated disease regression (Ferreri et al., J. Clin Oncol, 2005). To assess whether Chlamydia psittaci is associated with ocular adnexal lymphomas in a North American population, we studied 24 ocular adnexal lymphoma biopsy specimens from adults, 16 control lymphoma specimens from patients with systemic lymphomas not involving the ocular adnexa and five benign control ocular tissue samples. There were fifteen MALT, five diffuse, large B-cell, three follicular, and one mantle cell lymphoma specimens. The presence of Chlamydia psittaci DNA was investigated by PCR in each group. DNA was isolated from sections of formalin-fixed, paraffin-embedded tissue. Two different assays were utilized: 1) a previously published conventional PCR/gel based assay targeting a 111 bp fragment of the 16S gene [Madico, J. Clin Micro, 2000] and 2) a realtime PCR assay amplifying a 148 bp portion of the 16S gene with detection via a specific fluorescent probe. Amplification was carried out to 60 cycles. Positive controls consisted of isolated DNA from Chlamydia psittaci strain VS1, CP3, and FP. A human DNA internal control was utilized to assess DNA quality and amplification success. Both assays detected Chlamydia psittaci DNA from isolated strains. All patient samples in all categories yielded negative results using both assays. Thus, we could not confirm that Chlamydia psittaci is associated with ocular adnexal lymphomas in our patient population. Differences in the prevalence of Chlamydia psittaci infection in North America and Europe or technical differences in application of the assays may underlie the differences in our findings when compared to the Italian series. Confirmation with larger numbers of specimens is required.
Quantification of ADAMTS-13 Activity and Antigen from the Same Sample by a Newly Developed Combi-Actibind Assay (TECHNOZYM® ADAMTS-13).
Authors: Geiter Sabine , Colle Sebastién , Kyrle Paul A. , Scheiflinger Fritz , Binder Bernd R. , Vetr Helga
Published: April 2020

Defects in activity and/or antigen levels of ADAMTS-13, the von Willebrand Factor (vWF) cleaving protease, are viewed as the main cause inducing the microvascular thrombotic disorder TTP (thrombotic thrombocytopenic purpura) that is in more than 90% of cases fatal if not treated early and appropriately. Malfunction of ADAMTS-13 with respect to cleave multimeric vWF can be caused by auto-antibodies directed against ADAMTS-13, by decreased presence of ADAMTS-13 in the circulation or by defective activity of ADAMTS-13. Therefore rapid and reliable diagnosis of ADAMTS-13 parameters is a clinical need. We present here a new assay for quantification of ADAMTS-13 activity and antigen levels based on ELISA format and thus suitable for fast and routine measurements.

The method is based on the Actibind® technology, using a monoclonal antibody to capture ADAMTS-13 from plasma to a microtiter plate. Activity is then assayed by a fluorescent substrate. After removal of the substrate solution, antigen can be assayed by standard ELISA technology using a polyclonal antibody followed by a peroxidase labelled secondary antibody. Thus both parameters can be measured from the same sample consequently in the same assay allowing calculation of specific activities of ADAMTS-13.

Dilutions of normal plasma (pool of 100 normal donors) were used as calibrators. The standard curve for the activity assay comprises a range from 3.7% to 100 % of normal plasma activity, thus samples of severe TTP (definition: activity below 5%) can be detected with sufficient sensitivity. 42 samples from TTP patients were tested in this new activity/antigen assay as well as in the TECHNOZYM ADAMTS-13 INH assay that allows determination of anti ADAMTS-13 auto antibodies. Using these two assays a significant negative correlation was found between ADAMTS-13 activity and the presence of autoantibodies, 82% of the samples with extremely low ADAMTS-13 activity (<5%) had high levels of autoantibodies. A less stringent positive correlation was found between ADAMTS-13 antigen and ADAMTS-13 activity. Out of 30 samples with low ADAMTS-13 activity (< 25%) 15 samples had antigen levels less than 50% of normal. Out of the 42 samples from TTP patients, 3 samples were found without auto antibodies but still low activity. 2 of these samples also had low antigen levels. Moreover, the TTP samples had significantly higher levels of vWF:CBA activity (p<0.05) consistent with the presence of high molecular weight vWF.

This combined new assay clearly adds to the diagnostic possibilities to differentiate patients with TTP with respect to defects in ADAMTS-13 function and antigen concentration. In addition, the results of this assay correlate with the clinical picture and the decreased functional activity of ADAMTS-13 leading to increased levels of vWF multimers and in turn platelet consumption.
A Pathophysiological Role for ADAMTS13 in Thrombotic Thrombocytopenic Purpura (TTP) without Severe ADAMTS13 Deficiency.
Authors: Hovinga Johanna A. Kremer , George James N. , Vesely Sara K. , Terrell Deirdre R. , Budde Ulrich , Sulzer Irmela , Lammle Bernhard
Published: April 2020

Background: A severe ADAMTS13 deficiency (<5% of the normal) is a specific finding for an acute thrombotic microangiopathy commonly labeled as idiopathic TTP. However, the sensitivity of this finding for the clinical diagnosis of idiopathic TTP is only ~60% (range 33–100% in different studies) and the pathophysiology underlying idiopathic TTP in the absence of severe ADAMTS13 deficiency has not yet been elucidated.

Methods: 74 consecutive patients with acute acquired idiopathic TTP-HUS from the Oklahoma TTP-HUS registry (partly published by Vesely et al. [Blood2003;102:60–8]) were included into this study. For laboratory investigation, serum samples had been withdrawn before initiation of any form of treatment. ADAMTS13 activity was determined by quantitative immunoblotting, ADAMTS13 autoantibodies were evaluated by a routine inhibitor screening test and the TECHNOZYM ADAMTS13 INH ELISA, and patients’ VWF multimers were analyzed by SDS-agarose electrophoresis to assess endogenous VWF proteolysis arisen in vivo.

Results: An ADAMTS13 activity <5% was found in 22 (30%), an ADAMTS13 activity <10% in 28 (38%) patients. The ADAMTS13 INH ELISA gave positive results (≥15 arbitrary antibody U/ml) in 48 (65%) patients as well as in 15/113 (13.3%) controls. Setting a higher threshold of 35 arbitrary antibody U/ml (high titer), 33/74 (44.5%) patients and 3 (2.7%) controls showed positive results for autoantibodies to ADAMTS13. Out of these 33 patients, 28 had a positive inhibitor screening test, 24 had an ADAMTS13 activity <10%, and 25 displayed impaired or severely impaired VWF proteolysis on multimer analysis. In the intermediate antibody titer group (15-<35 U/ml) only 2/15 patients had an ADAMTS13 activity <10%, and 14/15 (93%) displayed normal or even increased VWF proteolysis. Of the remaining 26 (35%) patients without detectable ADAMTS13 autoantibodies, 2 (8%) had an ADAMTS13 activity <10%, and 22 (85%) had normal or even increased VWF proteolysis.

Conclusions: Most patients suffering from acute idiopathic TTP-HUS and high titers of ADAMTS13 autoantibodies had impaired VWF proteolysis in line with severely decreased ADAMTS13 activity. Patients without ADAMTS13 antibodies as well as the majority of patients with intermediate antibody titers demonstrated normal or even increased VWF proteolysis. (Severely) impaired proteolysis of endogenous VWF was observed in 3 patients with high titer anti-ADAMTS13 antibodies and normal ADAMTS13 activity (50–100%). Thus, our data suggest a pathophysiological role of ADAMTS13 in at least some patients despite normal ADAMTS13 activity by standard assay.
The Effect of Tumor Necrosis Factor (TNFα) Inhibition on Platelet Counts in Adults and Children with Persistent ITP.
Authors: Tarantino Michael D. , Stephens Dana M. , Imfeld Karen L. , Young Guy , Williams Shirley A. , Lucas Yvonne L. , Nugent Diane J.
Published: April 2020

Immune Thrombocytopenic Purpura (ITP) is hematologic disorder caused by autoimmune opsonization and premature destruction of platelets. Approximately 20% of children and the majority of adults will have a chronic course of ITP with antibody-mediated destruction of platelets and the inability to clear immune complexes. Pro-inflammatory cytokines IL-2, TNF-α and IFN-γ are secreted following a Th1 response and are elevated in patients with chronic ITP. Recent treatments for chronic ITP in adults and children have focused on quelling dysregulated T and B lymphocyte activity. We conducted a pilot clinical trial of etanercept, a fusion peptibody composed of the soluble TNF-α receptor and the Fc portion of Ig. Hematologic and immunologic responses were measured before, during (blood counts only) and after a 12-week regimen of 0.4mg/kg/dose (max dose 25 mg) of subcutaneous etanercept, given twice weekly. Only fixed-dose prednisone or danazol were allowed as concomitant ITP medications. Cytokine concentrations were measured pre- and post-treatment from plasma and cultured lymphocytes (unstimulated or PHA- [2.5ng/ml] stimulated). Sixteen patients (9 male, 7 female), with chronic ITP (8 post-splenectomy) an initial platelet concentration < 30 × 109/L were enrolled with data evaluable for 15. The mean age of the patients was 31 years (median 32, min/max 7/52 years). One patient withdrew due to dizziness thought related to the study drug. One patient developed autoimmune hemolytic anemia, was diagnosed with Evans syndrome at treatment week 3 and withdrew from the study. Otherwise, no severe or serious related adverse events were observed. Two patients had a complete platelet response (CR, plt count > 150 × 109/L at least 6 weeks post treatment), 6 had a partial response (PR, plt count > 30 × 109/L and double baseline plt count), one had a minimal response (MR, plt count > 30 × 109/L) and 5 had no platelet response (NR). Two of the patients with PR had a platelet count rise that lasted at least until the end of the 8 week post-treatment observation period. The platelet response rate in nonsplenectomized and splenectomized patients was 67% and 28%, respectively, p = 0.1. Concentrations of IL-1β, IL-4, IL-6, IL-10 IFN-γ and TGF-β, before and at the end of the 12-week treatment course, showed marked inter-patient variation and did not predict the platelet count outcome. Both patients that had a CR had a reduction in PHA-stimulated TNF-α of 33 and 30%, respectively. Others with NR to treatment had similar declines in TNF-α, however. Although the precise mechanism of action is unclear, etanercept treatment led to at least a partial platelet response in 50% of patients with persistent ITP and was well-tolerated.
An Unusual Form of Immune Thrombocytopenic Purpura Characertized by a Platelet Activating IgG Antibody.
Authors: Boyle Soames , Alexy Tamas , Rhochanda Leanne , Liebman Howard A.
Published: April 2020

Autoimmune thrombocytopenia (ITP) is classically a disorder of accelerated platelet clearance and ineffective platelet production due to antibodies directed against common platelet membrane glycoproteins. The most common clinical manifestation of severe thrombocytopenia is bleeding. We report an unusual case of ITP in a 49 y.o. female with a 4 year history of thrombocytopenia, venous and arterial thrombosis secondary to a platelet activating IgG antibody.

The patient was referred to one of the authors (HAL) for a second opinion with a diagnosis of ITP and a history of venous and arterial thrombosis. On presentation the patient was noted to have a platelet count of 34 × 109/L, a decreased fibrinogen of 128 mg/dL and D-dimers of greater than 4000 ng/ml. A diagnosis of chronic DIC was made and the patient was treated with therapeutic doses of LMW heparin resulting in an increase in her fibrinogen to 260 mg/dL, reduction in her D-dimers to <500 ng/ml and an increase in her platelet count to 118 × 109/L. An extensive clinical and laboratory evaluation, included screening for occult malignancy, cavernous hemangioma, antiphospholipid antibodies, dysfibrinogenemia, inhibitory antibodies against protein C and S, failed to disclose an etiology for her chronic consumptive coagulopathy. After 2 months of LMWH therapy the patient had a progressive drop in her platelet count to <30 × 109/L without evidence of recurrent coagulopathy. The patient’s thrombocytopenia was refractory to corticosteroids, Rituxan, azathioprine, cyclosporine, but responsive to IVIG. When the patients LMW heparin was stopped, the patient had evidence of a recurrence of her DIC as defined by an increase in her D-dimer and decreased fibrinogen. The patient underwent an open splenectomy which was complicated by portal vein and superior mesenteric vein thrombosis requiring catheter directed fibrinolysis and reinstitution of LMW heparin therapy. Repeated ELISA and functional assays for heparin induced thrombocytopenia were negative. However, the patient’s heat inactivated plasma and serum were shown to induce spontaneous aggregation. Patient IgG was subsequently isolated by staph A chromatography and found to be a potent inducer of platelet aggregation. Platelet specific IgG was further isolated from the patient’s IgG by affinity chromatography on a column of solubilized platelet membrane proteins. The affinity isolated antibody was then analyzed by the Western blotting using solubilized platelet membranes. Patient antibody was found to react with a 50 kD platelet membrane protein which has yet to be fully characterized.

At the present time the patient remains on LMW heparin, but requires frequent infusions of IVIG to maintain platelet counts above 30 × 109/L. We are aware of only one other reported case of a spontaneous platelet activating antibody and no other case characterized by chronic DIC.
Congenital Amegakaryocytic Thrombocytopenia in a Thrombocytopenic Child Presenting without Amegakaryocytosis.
Authors: Geddis Amy , Fox Norma , Nguyen Hung , Keates-Baleeiro Jennifer , Frangoul Haydar
Published: April 2020

Congenital amegakaryocytic thrombocytopenia (CAMT) is an inherited bone marrow syndrome due mutation of the thrombopoietin receptor c-Mpl. Affected children present with thrombocytopenia at birth and absence or severe reduction of megakaryocytes in the bone marrow and usually progress to complete bone marrow failure within the first decade of life. Mutations of c-Mpl have been classified as either type I mutations, in which the receptor has lost all activity, or type II mutations, in which the receptor retains some degree of function. Clinically, type II patients have a slightly delayed onset of bone marrow failure (mean age 48 months) compared to type I patients (22 months). Here we describe a girl with CAMT who had a clinical course similar to patients with type I mutations but without marked amegakaryocytosis at onset. The patient was born with intracranial hemorrhage due to severe thrombocytopenia (platelets 17K), which was initially thought to be due to alloimmune thrombocytopenia because of consistent platelet antigen typing studies and because the child responded to transfusion with her mother’s platelets and not to random donor platelets. However, thrombocytopenia persisted and she did not respond to immune directed therapies such as IVIG, prednisone or Rituximab, and after 14 months of age she began to develop neutropenia and anemia and by 24 months she had progressed to severe aplastic anemia. Bone marrow evaluation at 3 months showed trileage hematopoiesis with only mildly decreased megakaryocytes although they were noted to be small with hypolobulated nuclei. By 6 months of age, however, megakaryocytes appeared mildly decreased but were morphologically unremarkable and the marrow showed increased hematogones. At 24 months, marrow cellularity had decreased to 5% of normal with frank amegakaryocytosis. Sequencing of c-Mpl revealed two heterozygous mutations, one at Arg102Pro in the extracellular domain and the other one resulting in a stop codon at amino acid 541 in the intracellular domain. Arg102Pro is the most commonly occurring mutation reported in CAMT and is generally associated with type II disease. 541Stop is the first mutation reported in exon 11 and results in truncation of the receptor shortly after the box 1 homology domain. Previous in vitro studies involving the murine homolog of this mutation, which is missing all but the proximal 28 amino acids of the intracellular domain, demonstrated that it does not signal in response to thrombopoietin. This case illustrates the variation in clinical phenotype that can be seen in CAMT and the importance of gene sequencing for accurate diagnosis.
Thrombocytopoiesis Is Not Regulated by Thrombopoietin (TPO) but Proplatelet Formation (PPF) Stimulating Factor.
Authors: Murai Kazunori , Kowata Shugo , Tsukushi Yasuhiko , Ishiguro Mamiko , Oyake Tatsuo , Sugawara Takeshi , Ishida Yoji
Published: April 2020

TPO is believed to be the physiological humoral hematopoietic factor in megakaryopoiesis and thrombopoiesis. Some investigators reported the inverted relationship between platelet mass and serum TPO levels, resulting in the conclusion that the platelet mass may directly play a role of regulating the circulating TPO levels. In the previous ASH meeting, we reported that serum TPO levels were not elevated in Immune thrombocytopenic purpura (ITP) patients. In this study, we measured platelet count, the serum TPO level, % reticulated platelet (%RP) and megakaryocytes mass in patients with refractory anemia in myelodysplastic syndrome (MDS-RCMD). The mean plasma TPO level in 20 healthy persons was 57.3±15.2 pg/ ml, the percentage of RPs was 1.51±0.71%.

Plasma TPO levels were widely distributed (799.9±883.0 pg/ ml, n=35) in MDS-RCMD, while they were less than 131.0 pg/ml in ITP (48.5±37.0 pg/ ml, n=37). There were no significant relationship between platelet counts and plasma TPO levels in MDS-RCMD or ITP. These results indicated that plasma TPO level was not regulated by the platelet mass at least in thrombocytopenic patient with MDS-RCMD or ITP. We evaluated the thrombopoiesis in thrombocytopenic patients with MDS-RCMD or ITP. %RPs were 1.9±0.2 % in MDS-RCMD (n=34), indicating that increased thrombopoiesis was not observed in MDS-RCMD, in which serum TPO levels were increased. While %RPs were 6.6±4.9 % in ITP (n=45), indicating that thrombopoiesis was observed in ITP, in which serum TPO levels were not increased. These results indicated that thrombopoiesis was not regulated by plasma TPO levels but some thrombopoiesis stimulating factor(s). Therefore, we evaluated the thrombopoiesis stimulating activity in plasma of patients with MDS-RCMD or ITP as the indicator of PPF stimulating activity of murine megakaryocytes. PPF stimulating activity was measured as the ratio of PPF bearing megakaryocytes/ viable megakaryocytes after 48 hrs serum-free culture of murine megakaryocytes.

As the plasma have some inhibitory effects of PPF activiy, the HDL fraction, isolated from plasma, was used as the methods described previously in Ishida Y et al. (Thrombosis and Haemostasis, 2001; 85:349–55).

The higher PPF stimulating activity was observed in patients with ITP (n=15) or MDS-RCMD (n=15) than that with healthy donors (n=10) {healthy donors 26.1±5.4 % versus ITP 38.0± 6.0% (p<0.01), MDS-RCMD 41.9±5.3%(p<0.01)}. There was a significant correlation between the PPF activity and platelet counts (R=0.68; p<0.01) in these thrombopenic patients. These data strongly suggest that thrombopoiesis may not be regulated by TPO but some PPF stimulating factor(s).
Lenalidomide and CC-4047 Inhibit the Proliferation of Namalwa Cancer Cells While Expanding CD34+ Progenitor Cells. New Insights on the Combination Therapy with Certain HDAC Inhibitors for Hematological Cancers.
Authors: Verhelle Dominique , Corral Laura G. , Wong Kevin , Mueller Jessica H. , de Parseval Laure Moutouh , Jensen-Pergakes Kristen , Glezer Emilia , Ferguson Gregory D. , Brady Helen A.
Published: April 2020

Clinical studies involving patients with Myelodysplastic Syndrome and Multiple Myeloma have demonstrated the efficacy of lenalidomide (CC-5013), a drug recently approved by the FDA under the commercial name of Revlimid®, by reducing and often eliminating malignant cells while restoring bone marrow function. To better understand these clinical observations, we investigated and compared the effects of lenalidomide, and its analog CC-4047, on the proliferation of two different hematopoietic cell models: the Namalwa cancer cell line and CD34+ progenitor cells. We found that both compounds have anti-proliferative effect on Namalwa cells and pro-proliferative effect on CD34+ cells while p21WAF−1 expression was upregulated in both cell models. In Namalwa cells, we determined that p21WAF−1 is responsible for the inhibition of CDK2, CDK4, and CDK6 activity leading to pRb hypophosphorylation and cell cycle arrest. In contrast, in CD34+ progenitor cells, despite upregulated p21WAF−1 expression, we observed an increase of the cell division rate, leading to the enhancement of CD34+ expansion. Finally, we found that CC-4047 and lenalidomide have synergistic effects with two different HDAC inhibitors (Valproic acid and Trichostatin A) in both increasing the apoptosis of Namalwa cells and enhancing CD34+ cell expansion. Taken together, our results indicate that lenalidomide and CC-4047 have opposite effects in tumor cells versus progenitor cells and could explain, at least in part, the reduction of malignant cells and the restoration of the bone marrow observed in patients undergoing lenalidomide treatment. Moreover, this study provides new insights on the cellular pathways affected by lenalidomide and CC-4047, and proposes new potential clinical uses such as bone marrow regeneration. Finally, our vitro experiments showing the efficacy of the combination of CC-4047 and lenalidomide with Valproic acid and Trichostatin A suggest that HDAC inhibitors might be ideal candidates for combination therapy by elevating the therapeutic index, versus monotherapy, to treat hematological malignancies.
Prolonged High-Level Detection of Retrovirally Marked Hematopoietic Cells in Nonhuman Primates after Transduction of CD34+ Progenitors Using Clinically Feasible Methods.
Authors: Wu Tong , Kim Hyeoung Joon , Sellers Stephanie E. , Meade Kristin E. , Agricola Brian A. , Metzger Mark E. , Kato Ikunoshin , Donahue Robert E. , Dunbar Cynthia E. , Tisdale John F.
Published: April 2020

Low-level retroviral transduction and engraftment of hematopoietic long-term repopulating cells in large animals and humans remain primary obstacles to the successful application of hematopoietic stem cell(HSC) gene transfer in humans. Recent studies have reported improved efficiency by including stromal cells(STR), or the fibronectin fragment CH-296(FN), and various cytokines such as flt3 ligand(FLT) during ex vivo culture and transduction in nonhuman primates. In this work, we extend our studies using the rhesus competitive repopulation model to further explore optimal and transduction in the presence of either preformed autologous STR or immobilized FN. Long-term clinically relevant gene marking levels in multiple hematopoietic lineages from both conditions were demonstrated in vivo by semiquantitative PCR, colony PCR, and genomic Southern blotting, suggesting that FN could replace STR in ex vivo transduction protocols. Second, we compared transduction on FN in the presence of IL-3, IL-6, stem cell factor(SCF), and FLT(our best cytokine combination in prior studies)with a combination of megakaryocyte growth and development factor(MGDF), SCF, and FLT. Gene marking levels were equivalent in these animals, with no significant effect on retroviral gene transfer efficiency assessed in vivo by the replacement of IL-3 and IL-6 with MGDF. Our results indicate that SCF/G-CSF-mobilized PB CD34+ cells are transduced with equivalent efficiency in the presence of either STR or FN, with stable long-term marking of multiple lineages at levels of 10–15% and transient marking as high as 54%. These results represent an advance in the field of HSC gene transfer using methods easily applied in the clinical setting.
Structure of Erythropoietin in African Clawed Frogs, Xenopus laevis, and Its Role in the Liver Erythropoiesis.
Authors: Nogawa Nami , Kosaka Nobuyoshi , Aizawa Youichi , Miyazaki Hiroshi , Komatsu Norio , Kato Takashi
Published: April 2020

Erythropoietin (EPO) is a main regulator of erythropoiesis ensuring oxygen supply in mammalian species. However the functions of EPO in nonmammalian vertebrates remain unclear. In this study, EPO was identified in Xenopus laevis (X. laevis), and its contribution to definitive erythropoiesis was studied. The X. laevis EPO (xlEpo) cDNA revealed that the deduced amino acid sequence had only 38% identity to human EPO (hEPO), while all four cysteine residues were conserved. xlEPO mRNA was expressed predominantly in the liver and lung. In order to assess the biological activity, recombinant xlEPO was produced by transfecting COS-1 with CMV promotor-driven vector. A mouse FDC/P2 cells stably expressing xlEPOR cDNA, that is a putative EPO receptor, showed proliferation in response to recombinant xlEPO in a dose dependent manner. This confirmed the ligand-receptor relationship of nonmammalian xlEPO and xlEPOR. To our surprise, xlEPO stimulated proliferation of EPO-dependent human cell line UT-7/EPO as well as murine EPOR expressing FDC/P2 cell lines. The cross-reactivity suggests the tertially structure is conserved through xlEPO to mammalian EPOs. In addition, the amino acid residues that are essential for hEPO binding to hEPOR are highly conserved in xlEPO. Since potent N-glycosylation site is absent in xlEPO, the glycosylation characteristics of recombinant xlEPO was studied by fractionation using wheat germ aggulutinin (WGA) and concanavalin A (ConA) lectin affinity chromatography. XlEPO activity was seen in flow-through fractions indicating the absence of O-glycosylation as well as N-glycosylation in xlEPO molecule. The absence of glycosylation suggests the high affinity of xlEPO to xlEPOR, and the shorter blood half-life. In order to investigate the biological function of xlEPO, in vitro colony forming assay of X. laevis erythroid progenitors was developed. Magnetic cell sorting analysis showed that xlEPOR-positive cells reside in the liver possessing typical erythroblastic morphology with high nucleus-to-cytoplasm ratio containing hemoglobin. The formation of erythroblast colonies from liver cells on addition of recombinant xlEPO was observed. The colonies formed were erythroblast colonies composed of hemoglobin-synthesising erythroblasts, confirming the erythropoietic function of xlEPO in X. laevis erythropoiesis. These results and the detection of xlEPO mRNA in liver hypothesized the paracrine regulation of xlEPO. In the colony assay, erythropoietic activity was observed in the serum of phenylhydrazine (PHZ) induced anemic X. laevis. The highest erythropoietic activity was observed 4 days after PHZ-administration, prior to the peripheral erythrocyte number reaches a nadir at day 8. These results proved that xlEPO is a functional ortholog of mammalian EPO and its role in vertebrate hematopoietic system, providing new insights into the basis of erythropoietic regulations.
NE-180, a Novel glycoPEGylated Erythropoietin Demonstrates Dose-Dependent Activity in a Phase 1 Single Dose, Dose Escalating Study in Normal Human Volunteers (NHV).
Authors: Wallin Bruce A. , Ramjit Denise , Seiberling Michael , Zopf David
Published: April 2020

NE-180 is a glycoPEGylated recombinant human erythropoietin that binds to and activates the erythropoietin (EPO) receptor. It has demonstrated in vitro activities comparable to EPO and an extended serum half-life in animal studies. This may allow less frequent dosing in patients being treated with chronic anemia.

METHODS: A single center, open-label study of NE-180, administered as single escalating doses given by the SC or IV route, was conducted to assess the safety, tolerability, PK and PD. Subjects (male or female NHV) were planned to be assigned to one of 4 dose groups, 10 subjects per dose with 5 SC and 5 IV subjects per group: 0.5, 1.5, 3, or 4.5 mg/kg. Each dose group was planned to be initiated in an ascending, sequential fashion unless or until stopping rules were met.

RESULTS: 25 NHV (16 females) were enrolled in the first two dose cohorts and have completed 56 day follow-up. The 1.5 mg/kg IV cohort met the protocol-specified Hb rate of rise stopping rule (change in Hb greater than 1 g/dL during any 14 day period). Injections were generally well tolerated with no discontinuations for adverse events or serious adverse events. Reticulocyte increases were dose proportional. Average reticulocyte count at baseline was 1.0±0.3%. The maximal increase occurred at day 7. The mean change from baseline for the 0.5 and 1.5 mg/kg SC group was: 0.9±0.4% and 2.2±0.9%, respectively. The mean change from baseline for the 0.5 and 1.5 mg/kg IV group was: 1.7±0.8% and 2.3±0.8%, respectively. PK data will be presented.

CONCLUSIONS: Single doses up to 1.5 mg/kg of NE-180 administered to NHV were generally well tolerated and demonstrated potent dose-dependent erythropoietic activity.
Macrophage-Stimulating Protein Stimulates Erythropoietin Receptor Phosphorylation To Promote Enhanced Erk1/2 and Akt Activation in a Gab2-Dependent Pathway.
Authors: Chohan Manprit , Halupa Adrienne , Barber Dwayne L.
Published: April 2020

Friend-virus mediated erythroleukemia is a well-established model of murine oncogenesis. Several host factors have been shown to be necessary for Friend-mediated infection including a productive interaction between the Erythropoietin Receptor (EPO-R) and the 55 kDa glycoprotein product of the retroviral envelope gene (gp55). The Fv2 locus has been mapped and shown to be a truncated form of the STK tyrosine kinase (Sf-STK). Sf-STK expression and kinase activity is required for Friend virus infection of sensitive mouse strains. While the EPO-R and Sf-STK are required for Friend disease progression, it is unknown whether Sf-STK modulates EPO-R mediated signaling. EPO-R co-immunoprecipitated Sf-STK and STK specifically in COS-7 and Ba/F3 cells expressing both receptors. While the association between EPO-R and STK was shown to be constitutive, intriguingly, we discovered that co-stimulation with the STK ligand, Macrophage-Stimulating Protein (MSP) and EPO resulted in enhanced tyrosine phosphorylation of the EPO-R, compared to stimulation with EPO alone. Interestingly, STK kinase activity was required for EPO-dependent STK tyrosine phosphorylation, but was not required for MSP dependent EPO-R phosphorylation. Analysis of downstream signaling revealed STK and EPO-R co-operated in ligand induced dose-dependent phosphorylation of Erk1/2 and Akt. Interestingly, MSP and EPO also co-operated to stimulate tyrosine phosphorylation of the adaptor protein Gab2, leading to the recruitment of Shp2 and the p85 subunit of Phosphatidylinositol 3 kinase. These data suggest that Gab2 can couple to enhanced phosphorylation of Erk1/2 and Akt. However, no synergy was observed in MSP co-stimulation of JAK2, STAT1, STAT5, Jnk or p38 phosphorylation, demonstrating that STK activates specific EPO-dependent signaling pathways. EPO-dependent regulation of Socs family members was examined in Ba/F3-EPO-R cell lines expressing various STK constructs. The presence of STK induced Cis and Socs3, but not Socs1, transcript levels by 2.0-fold upon EPO stimulation. STK multi-functional docking sites (Y1330/Y1337) were required to observe optimal induction of Socs3. In contrast, Cis induction required STK tyrosine kinase activity. Our data demonstrate that EPO-R and STK interact at a biochemical level leading to augmentation of downstream signaling. These data suggest that Sf-STK contributes to Friend disease through delivery of mitogenic and/or survival signals to target erythroblast progenitor cells.