Background Coronavirus Disease 2019 (Covid-19) requiring hospitalization is characterized by robust antibody production, dysregulated immune response and immunothrombosis. Fostamatinib, is a novel spleen tyrosine kinase inhibitor we hypothesize will ameliorate Fc activation and attenuate harmful effects of the anti-COVID-19 immune response. Methods We conducted a double-blind, randomized, placebo-controlled trial in hospitalized adults requiring oxygen with Covid-19 where patients receiving standard of care were randomized to receive fostamatinib or placebo. The primary outcome was serious adverse events by day 29. Results A total of 59 patients underwent randomization (30 to fostamatinib and 29 to placebo). Serious adverse events occurred in 10.5% of patients in the fostamatinib group compared to 22% in placebo (P = .2). Three deaths occurred by day 29, all receiving placebo. The mean change in ordinal score at day 15 was greater in the fostamatinib group (-3.6 ± 0.3 vs. -2.6 ± 0.4, P = .035) and the median length in the ICU was 3 days in the fostamatinib group vs. 7 days in placebo (P = .07). Differences in clinical improvement were most evident in patients with severe or critical disease (median days on oxygen, 10 vs. 28, P = .027). There were trends towards more rapid reductions in C-reactive protein, D-dimer, fibrinogen and ferritin levels in the fostamatinib group. Conclusion For COVID-19 requiring hospitalization, the addition of fostamatinib to standard of care was safe and patients were observed to have improved clinical outcomes compared to placebo. These results warrant further validation in larger confirmatory trials.
Cancer stem cells have been identified in primary tumors, patient derived xenografts, and established cancer cell lines. The development of reporters has enabled investigators to rapidly enrich for these cells and more importantly track these cells in real time. Here we describe the current state of the reporter field and their use and limitations in multiple cancers.
Background Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by telangiectasias on mucocutaneous surfaces and arteriovenous malformations in visceral organs. Most patients present with epistaxis and gastrointestinal (GI) bleeding requiring iron supplementation and blood transfusion. There is no accepted medical treatment for HHT, although several studies suggest the potential efficacy of thalidomide. However, thalidomide is not available for treatment of patients with HHT. To address this, we initiated a Phase I trial of pomalidomide, a third generation thalidomide analogue with a lower incidence of cytopenias and neuropathy than thalidomide, in patients with HHT and dependence on iron infusion or blood transfusion. The aim of this phase 1 trial is to investigate the safety and efficacy of pomalidomide in HHT related epistaxis and GI bleeding. Methods Adult HHT patients with GI bleeding requiring at least 4 units transfusion or four doses of intravenous iron in the preceding four months, and patients with epistaxis requiring 2 units transfusion or 500 mg of intravenous iron in the preceding four months, and with an epistaxis severity score (ESS) ≥ 4 were enrolled. Pomalidomide was initiated at a 1 mg daily dose, which was increased by 1 mg/month to a maximal dose of 5 mg/day if bleeding did not entirely stop as judged by clinical evaluation. If bleeding stopped, patients were maintained on the dose of pomalidomide they were taking at the time of bleeding cessation for an additional 4 months. If bleeding did not stop, but improved, and patients escalated to the 5 mg/daily dose, they were maintained on this dose for an additional 4 months. After treatment with a stable dose of pomalidomide for 4 months, it was tapered by 1 mg/month until discontinued. The primary endpoint was a 50% reduction in the need for transfusion or parenteral iron therapy. A secondary endpoint was a reduction in the ESS of at least 1 point. Results The trial was designed for 9 patients and is continuing to accrue. Six patients, 5 males and 1 female, aged 48-70 with clinically diagnosed HHT provided informed consent. One patient withdrew consent before initiating therapy due to valvular heart disease requiring surgery. One patient had a drug-related adverse event (rash) soon after starting pomalidomide, which required removal from the study. Two patients (brothers) had non-drug related adverse events (diagnosis of a neuroendocrine tumor and recurrence of a perianal abscess requiring surgery) and were removed from the study within 5-7 months after initiation. One of these also had significant cramping after 7 months of treatment. Both of these siblings had presented with epistaxis and GI bleeding and reported a decrease in frequency of melanotic stools. Both required one course of IV iron soon after entering the study (prior to pomalidomide, they received iron every 2-3 months), however both maintained hemoglobin levels at least 1 gm higher than their previous baseline during study, and iron studies remained in the normal range after initiation of therapy. Two patients had primarily epistaxis. Both responded to pomalidomide within 1-2 months with decreases of > 50% (2-3 points) in the ESS; this was associated with an increase in quality of life. One of these patients required weekly iron infusions as well as red blood cell transfusions every 3-6 weeks before study; however, she has currently been on study for more than 6 months with a hemoglobin of 10-11 gm/dl, has not required blood transfusion, and has not received intravenous iron for more than 3 months with iron studies remaining normal. An additional patient has recently entered the study at the time of this abstract preparation. Pre- and post-treatment blood sampling has been performed and will be analyzed for biomarker changes prior to the ASH meeting. Toxicity has consisted primarily of grade 1-2 fatigue (n = 2), and cramps (n = 2), but these were not felt to be of sufficient severity to stop pomalidomide. Conclusions Preliminary interim findings from this phase I study provide an efficacy signal for patients with HHT and refractory bleeding. As with thalidomide, we speculate that pomalidomide causes remodeling of abnormal vasculature leading to decreases in AVMs and improvements in vessel stability. These findings support the development of a larger, multi-institutional HHT study. (NCT02287558) Disclosures No relevant conflicts of interest to declare.
Introduction Severe Aplastic Anemia (SAA) is a life-threatening bone marrow failure disorder associated with pancytopenia, serious infections, and transfusion dependence. Although long term survival for SAA patients (pts) can be achieved with immunosuppressive therapy (IST), one quarter to one third will fail to respond and about half of responders relapse. Many refractory SAA pts lack an HLA matched donor for salvage allogeneic stem cell transplantation. Although umbilical cord blood transplantation (UCBT) is an alternative approach for SAA pts, the procedure is associated with delayed engraftment and high rejection rates. Further, a substantial portion of UCB units have an insufficient number of TNCs/CD34+ cells for optimal transplant outcomes. To address the need to improve UCBT outcomes for SAA, we instituted a phase II trial exploring the use of nicotinamide (NAM) ex-vivo expanded UCB [omidubicel] to transplant pts with refractory SAA. Methods Eligible pts were 4-55 years and had transfusion dependent SAA. Other criteria included a) failure or intolerance to IST, b) lack of an HLA matched donor, c) having a ≥ 4/8 HLA matched UCB unit with a minimum of 1.8 x 109 and at least 1.8x107/kg TNCs and at least 8 x 106 CD34+ cells, and d) absence of donor specific antibodies to mismatched alleles on the UCB unit. The study was designed to enroll up to 6 pts receiving omidubicel and CD34+ selected haploidentical cells (cohort 1), followed by enrollment of up to 23 pts receiving omidubicel only (cohort 2). The conditioning regimen consists of hATG (40 mg/kg) on D -11 to -8, cyclophosphamide (60 mg/kg) on D -7 and -6, fludarabine (25 mg/m2) on D -5 to -1, and 2 Gy TBI on D -1. Tacrolimus and mycophenolate mofetil were given as GVHD prophylaxis. The primary end point of the study is sustained early engraftment. Secondary endpoints include treatment-related mortality, and standard transplant outcomes. Results A total of 8 pts with SAA refractory to IST and eltrombopag with a median age of 23 years (range 6-45) have been transplanted to date. Pts were heavily transfused (median ferritin 3745 µg/dL), had severe neutropenia with a median pre-transplant ANC of 115 (range 0-680), with 5 (63%) pts having HLA alloantibodies and 1 pt having an invasive fungal infection treated with multiple antifungals and granulocyte transfusions in the peri-transplant period. In cohort 1, the first 3 pts received omidubicel and ~3 x106 CD34+ cells/kg from a haploidentical donor. Since all 3 pts in cohort 1 had sustained engraftment with omidubicel, the study moved to cohort 2, where so far 5 pts have received omidubicel alone. UCB units were matched at a median of 5/8 HLA alleles (range 4-6). Before expansion, units contained a median 1.6 x105 CD34+ cells/kg. At time of transplantation, the expanded units contained a median 75.3 x105 CD34+ cells/kg, representing a median 42-fold expansion. With a median follow-up of 10 months (range 1-35), 7/8 (88%) pts have had early and sustained cord engraftment. One pt in cohort 2 failed to engraft and was salvaged with a haploidentical transplant. Another pt in cohort 2 died on D+ 62 from disseminated adenovirus infection. This pt also developed grade 2 acute GVHD and his IST was stopped early in an effort to boost immunity against adenovirus. The other 7 pts are alive without evidence for acute or chronic GVHD and are transfusion independent. CMV reactivation requiring treatment occurred in 3/6 (50%) of pts at risk. Neutrophil and platelet recovery have been remarkably quick, occurring at a median of 10 days (range 6-14) and 31 days (15-40) respectively. Among the 7 pts with sustained engraftment, six pts had ≥ 95% cord myeloid chimerism by D+ 14 and ≥ 95% T-cell chimerism by D+ 26. Immune recovery has also been remarkably brisk: at D+ 100, the median absolute CD4 count was 186/µL (IQR, 118-340) and mean (±SD) IgG level was 522(±161) mg/dL, respectively (Figure). Conclusions These results provide the first evidence that omidubicel can result in rapid engraftment and sustained hematopoiesis in heavily transfused and allo-immunized SAA pts who are at high risk for graft failure with conventional UCBT. Data showing rapid recovery of IgG levels and rapid reconstitution of CD4, CD8, and NK cell subsets suggests immune recovery may occur quicker with omidubicel compared to conventional UBCT. Taken altogether, our preliminary data suggest omidubicel represents a promising new alternative graft source for SAA pts who lack HLA matched donors. Figure Disclosures No relevant conflicts of interest to declare.
Background: Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder characterized by abnormal blood vessel formation, recurrent and severe epistaxis, gastrointestinal bleeding, as well as pulmonary, hepatic and cerebral arteriovenous malformation (AVM). The pathogenesis of HHT appears to involve dysregulation of TGF-β signaling in endothelial cells, though other mechanisms may also contribute. Elevated levels of VEGF have been described in some studies of HHT, though detailed molecular profiling of factors that regulate angiogenesis has not been performed, particularly before and after effective therapy. We have recently completed a pilot study of patients with HHT treated with pomalidomide that demonstrated safety and efficacy and suggested that pomalidomide may be a potential treatment option for this disorder (NCT02287558). Here, we describe the outcomes of this study and present correlative studies of potential biomarkers in the plasma of treated patients before, during and after treatment with pomalidomide. Methods: Eligible patients had 1) GI bleeding requiring transfusion of ≥ 4 units PRBC or 4 iron infusions, or 2) epistaxis with epistaxis severity score (ESS) ≥ 4 requiring ≥ 2 units PRBC or 500 mg intravenous iron, each within the preceding four months. The primary efficacy endpoint was a 50% reduction in parenteral iron infusion or blood transfusion. A key secondary endpoint was the effect of pomalidomide on the ESS. Treatment was initiated with 1 mg of pomalidomide daily, increasing by 1 mg/month to a maximum of 5 mg of daily. This dose, or a lower dose on which the patient had ceased bleeding was continued for four months and then tapered. The relative expression of 55 angiogenesis-related proteins was determined semi-quantitatively in the plasma of study subjects using the human angiogenesis proteome profiler array. In addition, the expression of seven angiogenesis-related proteins was determined quantitatively by electrochemiluminescence using a validated immunoassay (Meso Scale Discovery). Heparin-binding EGF-like growth factor (HB-EGF) levels were determined using a conventional sandwich ELISA. Results: Nine patients provided informed consent. One subject was not treated due to an intervening cardiac event. Two subjects developed a drug-related AE (rash) within three weeks of treatment initiation and were removed from the study, thus we report on 6 patients. Two subjects were on study for only 5 months and removed for non-drug related AEs; both had primarily GI bleeding. One of these met the primary endpoint while the other did not; however, this individual demonstrated a significant reduction in the ESS. The remaining four patients completed the study and all met the primary endpoint and demonstrated significant reductions in the ESS. Proteomic profiling of angiogenic biomarkers revealed a statistically significant basal elevation of vascular endothelial growth factor A (VEGF-A), VEGF-C, placental growth factor (PIGF), fibroblast growth factor-basic (FGF-basic) and heparin-binding EGF-like growth factor (HB-EGF) when compared to healthy, age matched controls. Levels of VEGF-D, soluble vascular endothelial growth factor receptor-1 (VEGFR1/Flt1), and tyrosine kinase-2 (Tie-2) exhibited a wide distribution at baseline, but overall were not significantly higher than those in healthy, matched controls. Responses to pomalidomide were associated with significant reductions of several angiogenic mediators including VEGF-A, VEGF-C, MMP8, MMP9, TIMP-4, Angiopoietin 1, and most notably HB-EGF. Conclusion: This pilot study conducted at the Cleveland Clinic HHT center provides demonstrates safety and efficacy of pomalidomide, a third generation thalidomide analogue, in patients with HHT. Differences in levels of several angiogenic mediators in patients HHT suggest a complex underlying pathogenesis with significant differences between individual patients. Angiogenic profiling indicates that pomalidomide treatment of HHT patients promotes the reduction of VEGF-A, VEGF-C, MMP8, MMP9, TIMP-4, Angiopoietin 1, and most notably HB-EGF, which may serve as potential biomarkers of pomalidomide efficacy. This study was supported by Celgene, Summit NJ Disclosures McCrae: Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Pomalidomide in Hereditary Hemorrhagic Telangiectasia
Introduction β2-glycoproteinI (β2GPI) is the primary antigen for antiphospholipid antibodies (Ab), and Ab to β2GPI are associated with thrombosis and recurrent fetal loss.β2GPIis comprised of 5 "sushi" domains. Complex disulfide bonding renders β2GPI a challenging protein to producerecombinantlyin high yield and most studies have utilized domain-deletion mutants produced on a lab scale for structure-function analyses.β2GPIalso has a complex tertiary structure, and is reported to circulate in a "circular" form that may "open" to expose the antigenic site for β2GPIAbunder specific conditions. We developed a novel method to produce recombinant β2GPI in which replacement of the leader peptide allows large scale expression using a lentiviral system with one-step purification on heparin-sepharose. The ability of this protein to bind anti-β2GPI Ab was compared with that of plasma-derived (wild type, WT) β2GPI. Methods β2GPIcDNAwas cloned into pLentiCMV DEST. The β2GPI containing vector was used to transduce HEK293 cells with selection using puromycin. β2GPIwas purified from conditioned medium using HiTrapHeparin HP. Plasma β2GPI was purified using a protocol employing perchloric acid precipitation followed by heparin-sepharose and Mono-S chromatography. To measure anti-β2GPI Ab, we analyzed plasma from 32 patients referred to the Cleveland Clinic Special Coagulation Laboratory for anti-β2GPI testing using the InovaQuanta-Lite ELISA. Normal plasma samples (n=15) were also analyzed at 1:100 dilution to determine cutoffs for anti-β2GPI positivity. Briefly, 96-well plates were coated overnight at 4° C with 2 µg/ml WT or recombinant β2GPI. After blocking β2GPI-coated plates with BSA, 50 µl of patient plasma at 1:10 and 1:100 dilutions were added to individual wells in quadruplicate. A standard curve for IgG binding to each plate was created using affinity-purified rabbit anti-β2GPI IgG at concentrations of 15, 31.25, 62.5, 125, and 250 ng/ml. After incubation for 30 minutes at room temperature, plates were washed three times and 100 µl of a 1:5000 dilution of goat anti-human IgG was added. After 30 minutes, wells were again washed prior to adding 100 µl/well of o-phenylenediamine dihydrochloride. Plates were read at 490 nm after 15 minutes following addition of 25 µl/well H2SO4. Results from different plates were standardized by extrapolating the amount of boundAb from the standard curve prepared on each plate. To compare performance of recombinant β2GPI against WT β2GPI in ELISA we first evaluated correlation using recombinant and WT β2GPI by Spearman's test. The two sets of ELISAs were also compared using the Wilcoxon matched pairs test. ELISA readings were considered positive if they were >90th percentile on a curve established using 15 normal plasmas. Sensitivity and specificity of the assays was determined with respect to the results of the clinical assay. Results Recombinant β2GPI was produced in high yield (10-20 mg/L) and purified to homogeneity with a single heparin chromatography step; the purified protein migrated as a single band of ~50 kDon SDS-PAGE with a characteristic increase in Mr upon reduction. Anti-β2GPI IgG ELISA using WT and recombinant β2GPI demonstrated excellent correlation at both 1:10 (Spearman's rho 0.70, P<0.001) and 1:100 dilution (Spearman rho 0.727, p<0.001) (Figure 1). Using the Wilcoxon test for paired samples, there was no significant difference between results of the ELISA using WT or recombinant β2GPI at 1:10 dilution (mean difference 4.26 ± 22.25, P<0.001) and a small difference at 1:100 dilution (mean difference 13.51 ±7.59, P <0.001) (Figure 2). Of the 32 patient samples, 6 were known positive for anti-β2GPI IgG (titer ≥ 20 GPL). Using a 90th percentile cutoff established using healthy volunteer samples, theELISA using recombinant β2GPI correctly identified 6/6 positive samples (sensitivity 100%). The ELISA using plasma-derived β2GPI correctly identified 5/6 positive samples (sensitivity 83.3%, specificity 84%). Conclusion Recombinant β2GPI can be produced in high yield by this novel method and purified with a single heparin chromatography step. It is recognized by anti-β2GPI Abat least as well as WT β2GPI. Further studies focused on site-directed mutagenesis of the intact molecule to optimize assays for detection of pathologic anti-β2GPI antibodies are underway. Disclosures No relevant conflicts of interest to declare.
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