Search

Autoimmune thrombotic thrombocytopenic purpura (aTTP) is caused by autoantibodies to the von Willebrand Factor cleaving enzyme, ADAMTS13. Despite recent advances in the treatment of acute aTTP, relapse rates remain high. Guidance for the treatment of patients in clinical remission but with persistent severe ADAMTS13 deficiency who fail to respond to rituximab remains unclear. We report a case of a 29-year-old man diagnosed with aTTP at the age of 11. Over a period of 18 years, he had five clinical relapses with persistent severe ADAMTS13 deficiency (

Thrombosis is known to occur in patients with rare inherited bleeding disorders, usually in the presence of a thrombotic risk factor such as surgery and/or factor replacement therapy, but sometimes spontaneously. We present the case of a 72-year-old African American male diagnosed with congenital factor VII (FVII) deficiency after presenting with ischemic stroke, presumably embolic, in the setting of atherosclerotic carotid artery stenosis. The patient had an international normalized ratio (INR) of 2.0 at presentation, with FVII activity of 6% and normal Extem clotting time in rotational thromboelastometry. He was treated with aspirin (325 mg daily) and clopidogrel (75 mg daily) with no additional bleeding or thrombotic complications throughout his admission. This case provides further evidence that moderate to severe FVII deficiency does not protect against thrombosis.

The standard dose of rituximab used in B-cell hematological malignancies, 375 mg/m2 weekly, may be excessive for autoimmune conditions. Successful use of a low, fixed dose of 100-200 mg of rituximab, weekly for 4 weeks, has been reported in the literature in the treatment of autoimmune thrombotic thrombocytopenic purpura (aTTP). We retrospectively analyzed our rituximab data in aTTP over a 13-year-period for 39 patients, with the aim of comparing response and outcomes with a standard lymphoma-dose course versus a low fixed 100 mg-dose course. Compared to the standard dose (17 patients, 17 courses of 4 infusions), our patients who received a low dose (8 patients, 9 courses of 4 infusions) had a possibly lower baseline risk but did achieve a similar time to remission and number of plasma exchange procedures to remission. Preemptive low-dose courses for ADAMTS13 activity

BACKGROUND Factor V (FV) deficiency may be inherited as an autosomal recessive disease or acquired as a result of autoantibody formation, either spontaneously or secondary to exposure to bovine thrombin or medications. Congenital FV deficiency has traditionally been treated with plasma transfusions. However, recent evidence has suggested that platelet (PLT) transfusions may be a better alternative as FV stored within PLT alpha granules has greater procoagulant potential and is released locally at sites of vascular injury. We report three cases of FV deficiency, one congenital and two acquired, and emphasize the different management approaches. CASE REPORTS Patient 1 was a 30-year-old man with congenital FV deficiency who presented with a trauma-induced hematoma of his lower extremity. He was treated with 5 PLT units over 48 hours. Patient 2 was a 64-year-old woman who presented with an upper-extremity thrombus and was discovered to have a FV inhibitor, likely secondary to antibiotics. Patient 3 was a 75-year-old woman with hepatitis C virus (HCV) who presented with minor ecchymosis and was found to have a FV inhibitor secondary to either HCV or antibiotic exposure. Corticosteroids alone were able to eradicate the inhibitors in both patients with acquired inhibitors. CONCLUSIONS FV deficiency can present with a diverse range of symptoms. For bleeding patients, PLT transfusions should be the initial therapy. In patients with thrombosis, the risks and benefits of anticoagulation must be carefully assessed before treatment. For patients with minor bleeds, transfusions may be withheld, and elimination of the inhibitor should be the primary objective.

Hypoxia-inducible factor-1 (HIF-1) is a key regulator of erythropoiesis. In this article, we report 3 novel mutations, P378S, A385T, and G206C, on the EGLN1 gene encoding the negative HIF-1α regulator prolyl hydroxylase domain-2 (PHD2) in 3 patients with isolated erythrocytosis. These mutations impair PHD2 protein stability and partially reduce PHD2 activity, leading to increased HIF-1α protein levels in cultured cells.

Heparin induced thrombocytopenia (HIT) is a serious adverse drug reaction caused by transient antibodies against platelet factor 4 (PF4)/heparin complexes, resulting in platelet activation and potentially fatal arterial and/or venous thrombosis. Most cases of HIT respond to cessation of heparin and administration of an alternative non-heparin anticoagulant, but there are cases of persisting HIT, defined as thrombocytopenia due to platelet activation/consumption for greater than seven days despite standard therapy. These patients remain at high risk for thrombotic events, which may result in limb-loss and mortality. Intravenous immunoglobulin (IVIg) has been proposed as an adjunct therapy for these refractory cases based on its ability to saturate FcγRIIa receptors on platelets, thus preventing HIT antibody binding and platelet activation. We describe 2 cases of persisting HIT (strongly positive antigen and functional assays, and persisting thrombocytopenia >7 days) with rapid clinical response to IVIg. We performed in-vitro experiments to support IVIg response. Healthy donor platelets (1 × 10e6) were treated with PF4 (3.75 μg/mL) for 20 min followed by 1-hour incubation with patients’ sera. Platelet activation with and without addition of IVIg (levels equivalent to those reached in a patient after treatment with 2 gm/Kg) was evaluated in the PF4-dependent P-selectin expression assay (PEA). A significantly decreased platelet activation was demonstrated after the addition of IVIg to both patient samples, which correlated well with the rapid clinical response that each patient experienced. Thus, our study supports the use of IVIg as an adjunct therapy for persisting HIT.

Background Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease resulting in systemic microvascular thrombosis. The disease is caused by excessive platelet (PLT) adhesion to ultra-large (UL) von Willebrand factor (VWF) multimers inadequately cleaved by the processing enzyme ADAMTS-13. While many cases respond to plasma exchange performed with or without concurrent corticosteroids, treatment of the 10% to 20% of patients with refractory disease is difficult. Experimental studies demonstrating that N-acetylcysteine (NAC) inhibits PLT binding to endothelial cell-secreted and anchored UL VWF multimers suggest that NAC may be useful in the treatment of TTP. Case report A 44-year-old woman presented with malaise, confusion, chest and abdominal pain, and transient visual loss. Laboratory results and peripheral blood smear were consistent with TTP. The patient was begun on plasma exchange and corticosteroid treatment, but after 10 days the PLT count was still less than 10.0 × 10(9) /L and she developed a fever. Rituximab was initiated, but the patient's condition worsened and she became comatose. Antibiotics were initiated, but cultures remained sterile. After 3 days of coma and further clinical deterioration, treatment with NAC was begun. The patient received a loading dose of 150 mg/kg NAC intravenously (IV) over 1 hour. Within 18 hours the patient awakened abruptly and began communicating with medical personnel. Plasma exchange, corticosteroids, rituximab, and NAC infusion (150 mg/kg IV over 17 hr daily × 10 days) were continued and by Day 17 the PLT count was more than 50 × 10(9) /L. The patient fully recovered and was discharged on Day 31. Conclusion This is the first complete report of a TTP patient treated with NAC. NAC was a safe and effective supplementary treatment for refractory TTP in this patient.

Prostate cancer remains a significant health problem for men in the Western world. Although treatment modalities are available, these do not confer long-term benefit and are accompanied by substantial side effects. Adoptive immunotherapy represents an attractive alternative to conventional treatments as a means to control tumor growth. To selectively target the tumor-expressed form of Muc1 we constructed a retroviral vector encoding a chimeric antigen receptor (CAR) directed against the aberrantly-expressed extracellular portion of Muc1 called the ‘variable number of tandem repeats’. We now demonstrate that T cells can be genetically engineered to express a CAR targeting the tumor-associated antigen Muc1. CAR-Muc1 T cells were able to selectively kill Muc1-expressing human prostate cancer cells. However, we noted that heterogeneous expression of the Muc1 antigen on tumor cells facilitated immune escape and the outgrowth of target-antigen loss variants of the tumor. Given the importance of androgen ablation therapy in the management of metastatic prostate cancer, we therefore also tested the value of combining conventional (anti-androgen) and experimental (CAR-Muc1 T cells) approaches. We show that CAR-Muc1 T cells were not adversely impacted by anti-androgen therapy and subsequently demonstrate the feasibility of combining the approaches to produce additive anti-tumor effects in vitro. Adoptive transfer of CAR-Muc1 T cells alone or in combination with other luteinizing hormone-releasing hormone analogs or antagonists should be tested in human clinical trials.

A previously healthy 44-year-old woman presented with 3 days of worsening petechial rash, epistaxis and fatigue. Admission labs revealed pancytopenia, low reticulocyte index and elevated liver enzymes. Bone marrow biopsy demonstrated a profoundly hypocellular bone marrow without dysplasia and additional testing demonstrated an acute hepatitis B infection. In the context of an acute hepatitis B infection, elevated liver enzymes and aplastic anaemia, our patient was diagnosed with severe hepatitis-associated aplastic anaemia due to an acute hepatitis B infection. She was initiated on antiviral therapy with tenofovir and briefly received immunosuppressive therapy with a robust sustained improvement in her blood counts. Acute hepatitis B-associated aplastic anaemia is an exceptionally rare presentation of aplastic anaemia. We present acute hepatitis B-associated aplastic anaemia that resolved with antiviral therapy, which to our knowledge is the second such case reported in the literature and the first using tenofovir.

Introduction: Acquired hemophilia A (AHA) is a rare (1.5 per million/year) autoimmune disorder due to formation of spontaneous antibodies against factor VIII (FVIII) with an estimated mortality of 8-22%. First-line hemostatic agents to control a bleed include activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rVIIa) whereas immunosuppressive therapy to eradicate the inhibitor include prednisone, cyclophosphamide, and rituximab. We present data on 12 AHA patients from our institution. Methods: We conducted a retrospective review of patients with AHA from January 1, 2010 to July 1, 2016. Results: Eight women and 4 men with a median age of 68 years (range 29-88 years) were included in the study. AHA was idiopathic in 7 patients, others being associated with HIV/Hepatitis C (1), Hepatitis C only (1), lymphoma (1) pregnancy (1), and pregnancy/systemic lupus erythematosus (SLE) (1). Of the 12 patients, 11 were admitted at least once with a bleeding episode. In total, there were 38 bleeding episodes, with 15 being characterized as severe (39%). The most common site of bleeding was deep muscle (50%), and 71% of bleeding episodes were considered spontaneous. The median FVIII activity at admission was Table 1 summarizes the number of bleeding episodes and immunosuppression for each patient. Patient 7 experienced 24 bleeding episodes with 23 requiring both aPCC (median 75 u/kg, range 50-80 u/kg) and rVIIa (median 50 mcg/kg, range 50-90 mcg/kg) alternating every 6 hours. This patient had breakthrough bleeds on either aPCC or rFVIIa but responded to the alternating regimen. Patient 11 developed severe bleeding in his upper extremities, necessitating bilateral upper extremity amputation. He was initially treated with only aPCC (100u/kg every 12 hours for 11 doses total); however, concern for disseminated intravascular coagulation prompted aPCC discontinuation. He received a total 121 doses of rVIIa (90mcg/kg every 2-4 hours) during his hospital stay. Patient 2 required two doses of rVIIa (90 mcg/kg every 12 hours) after having continued bleeding despite treatment with 11 doses of aPCC (50u/kg every 12 hours). Ten bleeding episodes were treated with only aPCC with a median 8 doses (range 2-16 doses), while 1 bleeding episode was controlled with only rVIIa. One bleeding episode did not require treatment. The median dosage of aPCC (with or without rVIIa) was 75u/kg (range 50-100 u/kg) given every 12 hours (range 8-24 hours) for 8 doses (range 1 -16 doses). The median dosage of rVIIa when used in conjunction with aPCC was 50mcg/kg (range 50-100 mcg/kg) given every 12 hours (range 4-24 hours) for 6 doses (range 1-13 doses). No patient experienced a thromboembolic event. Follow-up data was available for 9 patients (Table 1). Six patients achieved complete remission (CR) at a median 126 days (range 68-1103 days). Patient 12 was initially treated with 1mg/kg prednisone; however, upon tapering, her FVIII activity shortened from 114% to 36%. Cyclophosphamide (2mg/kg) was added with eventual inhibitor eradication. Patients 3 and 7 failed to attain CR and were treated with prednisone, cyclophosphamide, and rituximab. They continued to have a FVIII activity Conclusion: CR was achieved in 6 of 9 patients with follow-up data. There were no deaths related to AHA. A combination of aPCC and rVIIa, alternating every 6 hours, can safely be used to treat severe bleeding, reducing drug and administrative costs. Disclosures Sarode: CSL Behring: Consultancy, Honoraria.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease resulting in systemic microvascular thrombosis. The disease is caused by excessive platelet (PLT) adhesion to ultra-large (UL) von Willebrand factor (VWF) multimers inadequately cleaved by the processing enzyme ADAMTS-13. While many cases respond to plasma exchange performed with or without concurrent corticosteroids, treatment of the 10% to 20% of patients with refractory disease is difficult. Experimental studies demonstrating that N-acetylcysteine (NAC) inhibits PLT binding to endothelial cell-secreted and anchored UL VWF multimers suggest that NAC may be useful in the treatment of TTP.A 44-year-old woman presented with malaise, confusion, chest and abdominal pain, and transient visual loss. Laboratory results and peripheral blood smear were consistent with TTP. The patient was begun on plasma exchange and corticosteroid treatment, but after 10 days the PLT count was still less than 10.0 × 10(9) /L and she developed a fever. Rituximab was initiated, but the patient's condition worsened and she became comatose. Antibiotics were initiated, but cultures remained sterile. After 3 days of coma and further clinical deterioration, treatment with NAC was begun. The patient received a loading dose of 150 mg/kg NAC intravenously (IV) over 1 hour. Within 18 hours the patient awakened abruptly and began communicating with medical personnel. Plasma exchange, corticosteroids, rituximab, and NAC infusion (150 mg/kg IV over 17 hr daily × 10 days) were continued and by Day 17 the PLT count was more than 50 × 10(9) /L. The patient fully recovered and was discharged on Day 31.This is the first complete report of a TTP patient treated with NAC. NAC was a safe and effective supplementary treatment for refractory TTP in this patient.

Human 17β-hydroxysteroid dehydrogenase types 1 and 2 (17βHSD1 and 17βHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17βHSD1 and 17βHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17βHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2′-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP+ ratio. The shift to E2 oxidation parallels changes in apparent Km values for purified 17βHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17βHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17βHSD2, and these mutations lower the apparent Km of recombinant 17βHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17βHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP+ ratios. In contrast, the preference of 17βHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17βHSD2 in intact cells.

The hydroxysteroid dehydrogenases (HSDs) interconvert pairs of weak and potent steroids, thus serving as key enzymes in the regulation of intracellular hormone potency. These enzymes may appear to drive unidirectional steroid flux in intact cells but actually catalyze bi-directional metabolism that achieve pseudo-equilibria with strong directional preferences. Even small shifts in the magnitude of these pseudo-equilibria can profoundly change steroid potency and thus contribute to disease. Consequently, we are studying the structural and biochemical principles that govern these directional preferences and the resilience of these pseudo-equilibria in intact cells. HSD directional preferences in intact cells are governed largely by relative affinities for nicotinamide cofactors [NAD(P)(H)] and existing cofactor gradients. We can attenuate the directional preferences for human 17βHSD type 1 and rat AKR1C9 in intact cells by either diminishing the NADPH/NADP+ gradient or by mutating the arginine residues that form salt bridges with the 2′-phosphate of NADP(H) (R38 and R276, respectively).

Background Ten to thirty percent of patients with immune thrombocytopenia (ITP) do not respond to initial therapy. Options for such patients include Rituximab, TPO-mimetic agents and splenectomy. We report on three patients with refractory ITP and significant thrombocytopenia who received romiplostim as a bridge to splenectomy. Methods Three patients with isolated ITP or Evans syndrome did not respond to steroids, intravenous immune-globulin (IVIG) or Rituximab. Romiplostim was given to increase the platelet count prior to laparoscopic splenectomy. Before surgery the bone marrow was evaluated and patients were vaccinated. We reviewed their baseline clinical characteristics, and clinical course. Results Patient 1: 51 y/o M presented with fatigue and melena. Laboratory data showed hemoglobin (Hgb) of 7.9 g/dl, platelet (plt) count of < 2,000/uL, LDH of 997 U/L, reticulocyte count of 7.5 % and positive direct Coombs test. Prednisone and IVIG were started with improvement in Hgb but plt count remained < 2000/uL. Rituximab was started on day 7 with no response in plt count after 4 doses. Weekly romiplostim was started on day 37 with no response after 3 doses and then pt was lost to follow up. He presented two months later with plt count of < 2,000/uL and was treated with IVIG with transient response and restarted on Romiplostim. Plt count reached to 93,000/uL with a dose of 6 mcg/kg of romiplostim. He was maintained on romiplostim for a total of 19 doses and then underwent laparoscopic splenectomy. Platelet counts pre-op were 294,000/uL, post-op rose to 1,261,000/uL but normalized afterwards and he has been off all treatment for more than two years (figure 1). Patient 2: 28 y/o F presented with bruising and plt count of 12,000/uL. She was treated with 5 days of dexamethasone with transient improvement in plt count to 50,000/uL which rapidly dropped to < 2,000/uL. Despite daily prednisone and IVIG, plt count continued to be < 2000/uL and weekly rituximab was started on day 7. On day 14, plt count continued to be Patient 3: 66 y/o M with coronary artery disease and long standing ITP was on prednisone 10-20 mg/day with plt count of 10-20,000/uL. Because of side effects of steroids, he was treated with danazol and rituximab (6 weeks) with no response in plt count. Cardiac catheterization prior to splenectomy could not be done because of thrombocytopenia. Romiplostim was then started at dose of 1 mcg/kg and advanced weekly to 3 mcg/kg with improvement in counts to 60,000/uL with peak of 200,000/uL. Cardiac catheterization was normal and he underwent successful laparoscopic splenectomy. Plt count before splenectomy was 73,000/uL and increased to 513,000/uL post-op. The plt count decreased to 120,000/uL and has remained stable for more than two years without further treatment. Conclusions Rituximab and TPO-mimetic agents are increasingly used for treatment of relapsed, refractory ITP as an alternative to splenectomy. Rituximab has a significant relapse rate. TPO-mimetic agents are expensive, require lifelong use, and are associated with rebound thrombocytopenia in those who stop treatment. Long-term use has also been associated with reversible bone marrow fibrosis and thromboembolism. Laparoscopic splenectomy is a safe procedure that is ideally done after a platelet response to high dose steroids and/or IVIG to reduce bleeding risk. Two of our patients were refractory to Rituximab. One received Rituximab concurrently with Romiplostim. All 3 patients did not want long term treatment with a TPO agent and had low platelet counts that were an impediment to surgery. We found that a short course of Romiplostim increased the platelet counts enough to enable surgery. All 3 patients achieved sustained remissions requiring no further treatment. No rebound thrombocytopenia was observed after stopping Romiplostim and none of the patients had bleeding or thrombotic complications. This case series suggests that a short course of Romiplostim can be used safely and effectively as a bridge to splenectomy for patients with refractory ITP. Disclosures: No relevant conflicts of interest to declare.