Impaired fibrinolysis in JAK2V617F-related myeloproliferative neoplasms.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are characterized by a high rate of thrombotic complications that contribute to morbidity and mortality. MPN-related thrombogenesis is assumed to be multifactorial, involving both procoagulant and proinflammatory processes. Whether impaired fibrinolysis also participates in the prothrombotic phenotype of MPN has been poorly investigated. OBJECTIVES: We determined whether MPN, particularly JAK2V617F-positive MPN, is associated with fibrinolytic changes. METHODS: Tissue-type plasminogen activator (tPA)-mediated fibrinolysis was evaluated both in whole blood and plasma from mice with a hematopoietic-restricted Jak2V617F expression compared with wild-type (WT) mice (Jak2WT) using (1) halo clot lysis, (2) front lysis, and (3) plasmin generation assays. tPA clot lysis assay was performed in the plasma from 65 MPN patients (JAK2V617F mutation, n = 50; CALR mutations, n = 9) compared with 28 healthy controls. RESULTS: In whole blood from Jak2V617F mice, we observed a decreased fibrinolysis characterized by a significantly lower halo clot lysis rate compared with Jak2WT (95 ± 22 vs 147 ± 39 AU/min; P < .05). Similar results were observed in plasma (halo clot lysis rate, 130 ± 27 vs 186 ± 29 AU/min; front lysis rate, 2.8 ± 1.6 vs 6.1 ± 1.2 µm.min-1; P < .05). Plasmin generation was significantly decreased both in plasma clots and standardized fibrin clots from Jak2V617F mice compared with Jak2WT mice. Among MPN patients, impaired tPA-related fibrinolysis with prolonged clot lysis time was observed in JAK2V617F and CALR patients. Plasminogen activator inhibitor-1 and α2-antiplasmin were significantly increased in plasma from JAK2V617F patients compared with controls. CONCLUSION: Our results suggest that impaired tPA-mediated fibrinolysis represents an important prothrombotic mechanism in MPN patients that requires confirmation in larger studies.

Exacerbation of thromboinflammation by JAK2V617F mutation worsens the prognosis of cerebral venous sinus thrombosis.

ABSTRACT: Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for <1% of strokes resulting in brain parenchymal injuries. JAK2V617F mutation, the most frequent driving mutation of myeloproliferative neoplasms, has been reported to be associated with worse clinical outcomes in patients with CVST. We investigated whether hematopoietic JAK2V617F expression predisposes to specific pathophysiological processes and/or worse prognosis after CVST. Using an in vivo mouse model of CVST, we analyzed clinical, biological, and imaging outcomes in mice with hematopoietic-restricted Jak2V617F expression, compared with wild-type Jak2 mice. In parallel, we studied a human cohort of JAK2V617F-positive or -negative CVST. Early after CVST, mice with hematopoietic Jak2V617F expression had increased adhesion of platelets and neutrophils in cerebral veins located in the vicinity of CVST. On day 1, Jak2V617F mice had a worse outcome characterized by significantly more frequent and severe intracranial hemorrhages (ICHs) and higher mortality rates. Peripheral neutrophil activation was enhanced, as indicated by higher circulating platelet-neutrophil aggregates, upregulated CD11b expression, and higher myeloperoxydase plasma level. Concurrently, immunohistological and brain homogenate analysis showed higher neutrophil infiltration and increased blood-brain barrier disruption. Similarly, patients with JAK2V617F-positive CVST tended to present higher thrombotic burden and had significantly higher systemic immune-inflammation index, a systemic thromboinflammatory marker, than patients who were JAK2V617F-negative. In mice with CVST, our study corroborates that Jak2V617F mutation leads to a specific pattern including increased thrombotic burden, ICH, and mortality. The exacerbated thromboinflammatory response, observed both in mice and patients positive for JAK2V617F, could contribute to hemorrhagic complications.

Massive circulating metastatic cells: A case of carcinocythemia.

Importance of careful differential diagnosis to make the distinction between carcinocythemia and acute leukemia or lymphoma.

Heparin-induced thrombocytopenia in newborns: experience at a pediatric hospital and a literature review.

Background: Heparin-induced thrombocytopenia (HIT) is a rare, difficult-to-diagnose, and potentially serious adverse drug reaction with thrombotic complications. Even though the immune system is still immature during the neonatal period, HIT has been described in newborns with reporting rates ranging from 0% to 2.3%. Therefore, it is important to clarify the risk of HIT in newborns because it can affect the management and monitoring of heparin treatment. Objectives: The objectives of the present study were to review the literature and determine the incidence of HIT after cardiac surgery in newborns in our pediatric hospital. Methods: We searched the literature from 1992 to 2021 for reports of HIT in newborns. Four raters then analyzed all the literature reports on HIT and classified them as "likely," "uncertain," or "unlikely." We also determined the incidence of HIT among newborns having undergone cardiac surgery in our pediatric hospital. Results: Eleven population-based studies and 12 case reports on suspected HIT in 17 newborns were reviewed. One study reported HIT in 14 out of 930 (1.5%) heparin-treated newborns, but the other studies (n = 467 newborns) did not mention HIT at all. None of the cases described in the literature was classified as "likely" by the raters. In our center, none of the 2997 newborns that had undergone cardiac surgery in the previous 16 years was diagnosed with HIT. Conclusion: We conclude that the incidence of HIT in newborns has been overestimated in the literature.

State of the art in cerebral venous sinus thrombosis animal models.

Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for less than 1% of strokes resulting in brain parenchymal injuries. Diagnosis and prognosis are still challenging due to highly variable clinical course and etiologies. Beyond thrombosis, different CVST-related parenchymal injuries may occur and include edema, ischemic strokes, and intra-cerebral hemorrhage (ICH; i.e., parenchymal/subdural hematomas, and subarachnoid hemorrhages), which are identified in 40%-60% of patients without clearly identified mechanisms. In this perspective, experimental animal models contribute to the understanding of initiation, propagation, and resolution of thrombosis, as well as brain-related damages. Last but not least, animal models may be useful to study new therapeutic approaches. In this review, we provide a comprehensive overview of CVST experimental models, focusing on their strengths, limits, and contribution to the current knowledge.

PCR-Fluo-ASXL1-FA: A fast, sensitive and inexpensive complementary method to detect ASXL1 mutations in haematological malignancies.

INTRODUCTION: The additional sex combs like 1 (ASXL1) gene is frequently mutated in a number of haematological neoplasms. The c.1934dupG, known to be the most common alteration in ASXL1, is associated with poor clinical outcome. A systematic determination of ASXL1 mutational status in myeloid malignancies is therefore necessary for prognostic stratification. METHODS: Because direct sequencing is not sensitive and next-generation sequencing (NGS) is time-consuming, expensive and sometimes does not allow the detection of the c.1934dupG, we have developed a fragment analysis assay, complementary to NGS, that allows the detection of c.1934dupG mutation in addition to other nearby insertions/deletions of ASXL1 located close to it. We called this assay the "PCR-Fluo-ASXL1-FA." RESULTS: First, we evaluated the efficiency of our approach compared to NGS and Sanger. We showed that "PCR-Fluo-ASXL1-FA" could detect all insertional mutations of ASXL1 located on its area, with a high sensitivity (1.5%). Then, we have illustrated the interest of this technique by three concrete cases. DISCUSSION: In summary, we have established a fragment analysis approach, which can detect most ASXL1 mutations, in particular the c.1934dupG, in a sensitive, fast and inexpensive manner. We therefore recommend the synchronous use of this method with NGS, to ensure complete detection of all clinically relevant ASXL1 mutations in patients suffering with myeloid neoplasms.

A Novel Mouse Model for Cerebral Venous Sinus Thrombosis.

Cerebral venous sinus thrombosis (CVST) is an uncommon cause of stroke resulting in parenchymal injuries associated with heterogeneous clinical symptoms and prognosis. Therefore, an experimental animal model is required to further study underlying mechanisms involved in CVST. This study is aimed at developing a novel murine model suitable and relevant for evaluating injury patterns during CVST and studying its clinical aspects. CVST was achieved in C57BL/6J mice by autologous clot injection into the superior sagittal sinus (SSS) combined with bilateral ligation of external jugular veins. Clot was prepared ex vivo using thrombin before injection. On days 1 and 7 after CVST, SSS occlusion and associated-parenchymal lesions were monitored using different modalities: in vivo real-time intravital microscopy, magnetic resonance imaging (MRI), and immuno-histology. In addition, mice were subjected to a neurological sensory-motor evaluation. Thrombin-induced clot provided fibrin- and erythrocyte-rich thrombi that lead to reproducible SSS occlusion at day 1 after CVST induction. On day 7 post-CVST, venous occlusion monitoring (MRI, intravital microscopy) showed that initial injected-thrombus size did not significantly change demonstrating no early spontaneous recanalization. Microscopic histological analysis revealed that SSS occlusion resulted in brain edema, extensive fibrin-rich venular thrombotic occlusion, and ischemic and hemorrhagic lesions. Mice with CVST showed a significant lower neurological score on post-operative days 1 and 7, compared to the sham-operated group. We established a novel clinically CVST-relevant model with a persistent and reproducible SSS occlusion responsible for symptomatic ischemic and hemorrhagic lesions. This method provides a reliable model to study CVST physiopathology and evaluation of therapeutic new regimens.

Dabigatran Level Before Reversal Can Predict Hemostatic Effectiveness of Idarucizumab in a Real-World Setting.

Background: Idarucizumab has been included in guidelines for the management of bleeding or surgical procedure in dabigatran-treated patients without need for biological monitoring. The aim of the study was to assess the prognostic value of dabigatran plasma level before reversal to test the hemostatic efficacy of idarucizumab. The secondary objectives were (i) to analyze plasma dabigatran level according to the risk of rebound and (ii) to evaluate the incidence of post-reversal non-favorable clinical outcomes (including thromboembolism, bleeding, antithrombotic, and death) and antithrombotic resumption. Methods and Results: This was an observational multicentric cohort study, which included all French patients who required idarucizumab for dabigatran reversal. Between May 2016 and April 2019, 87 patients from 21 French centers were enrolled. Patients received idarucizumab for overt bleeding (n = 61), urgent procedures (n = 24), or overdose without bleeding (n = 2). Among patients with major bleeding (n = 57), treatment with idarucizumab was considered effective in 44 (77.2%) of them. Patients who did not achieve effective hemostasis after reversal had a significantly higher mean level of plasma dabigatran at baseline (524.5 ± 386 vs. 252.8 ng/mL ± 235, p = 0.033). Furthermore, patients who did not achieve effective hemostasis after reversal had less favorable outcomes during follow-up (46.2 vs. 81.8%, p = 0.027). ROC curve identified a cutoff of 264 ng/mL for dabigatran level at admission to be predictive of ineffective hemostasis. No plasma dabigatran rebound was observed after reversal in patients with dabigatran plasma level < 264 ng/mL at baseline. Conclusion: This retrospective study shows that dabigatran level before reversal could predict hemostatic effectiveness and dabigatran plasma rebound after idarucizumab injection.

Lupus anticoagulant diagnosis in patients receiving direct oral FXa inhibitors at trough levels: A real-life study.

INTRODUCTION: Direct oral factor Xa inhibitors (xabans) induce false positive results for lupus anticoagulant (LA) diagnosis. Consequently, it is suggested not to perform LA testing in xabans patients although it may be useful in selected patients. In this monocentric study, we evaluated xabans impact at trough levels (ie, just before the next intake) on LA diagnosis in treated patients using dilute Russell viper venom time (dRVVT) and two LA sensitive activated partial thromboplastin time (aPTT). METHODS: Sixty patients receiving rivaroxaban (30) or apixaban (30) were included. Plasma concentrations were measured using specific anti-Xa assays. LA testing was performed using one dRVVT (LAC-Screening® /Confirm® ; Siemens) and two LA sensitive aPTT-based assays (Hemosil® Silica Clotting Time (SCT) Screen/Confirm; Werfen and Dade® Actin® Factor Sensitivity FSL/FS (Actin F); Siemens). RESULTS: Median [min-max] concentrations were 23 [<18-68] for rivaroxaban and 42 ng/mL [19-99] for apixaban. dRVVT was positive in 93% of rivaroxaban and 40% of apixaban samples. SCT was positive in 40 and 30% and Actin F in 17 and 20% of samples respectively. Xabans affected more significantly dRVVT than aPTT-based assays (P < .001) with less false positive results with apixaban than with rivaroxaban samples irrespective of the assay used. CONCLUSION: lupus anticoagulant diagnosis in rivaroxaban and apixaban samples drawn at trough levels remains questionable whenever positive results are obtained. If LA testing in apixaban samples might be useful to rule-out LA using dRVVT and/or aPTT-based assays, the wide majority of rivaroxaban samples would give false positive results.

Disseminated toxoplasmosis associated with hemophagocytic syndrome after kidney transplantation: A case report and review.

Disseminated toxoplasmosis is infrequent after kidney transplant transmission but life-threatening because of a lack of diagnostic suspicion as well as specific chemoprophylaxis recommendations. Solid organ transplantation has resulted in few cases of disseminated toxoplasmosis presenting with associated hemophagocytic syndrome. Herein, we report, within the context of a donor/receiver mismatch, a case of a toxoplasmosis associated with hemophagocytic syndrome in a kidney transplant recipient. Molecular and serological investigations confirmed Toxoplasma gondii transmission through the kidney graft.

Phase 1 Clinical Trial of Trametes versicolor in Women with Breast Cancer.

Introduction. Orally administered preparations from the Trametes versicolor (Tv) mushroom have been hypothesized to improve immune response in women with breast cancer after standard chemotherapy and radiotherapy. Methods. A phase I, two-center, dose escalation study was done to determine the maximum tolerated dose of a Tv preparation when taken daily in divided doses for 6 weeks after recent completion of radiotherapy. Eleven participants were recruited and nine women completed the study. Each cohort was comprised of three participants given one of three doses of Tv (3, 6, or 9 grams). Immune data was collected pre- and postradiation, at 3 on-treatment time points and after a 3-week washout. Results. Nine adverse events were reported (7 mild, 1 moderate, and 1 severe), suggesting that Tv was well tolerated. Immunological results indicated trends in (1) increased lymphocyte counts at 6 and 9 grams/day; (2) increased natural killer cell functional activity at 6 grams/day; (3) dose-related increases in CD8(+) T cells and CD19(+) B cells , but not CD4(+) T cells or CD16(+)56(+) NK cells. Conclusion. These findings show that up to 9 grams/day of a Tv preparation is safe and tolerable in women with breast cancer in the postprimary treatment setting. This Tv preparation may improve immune status in immunocompromised breast cancer patients following standard primary oncologic treatment.

Trastuzumab and interleukin-2 in HER2-positive metastatic breast cancer: a pilot study.

PURPOSE: Trastuzumab as a single agent has activity in metastatic breast cancer; however, the mechanism of action for this clinical activity is uncertain. Whereas interruption of erbB family member signaling occurs, trastuzumab also mediates antibody-dependent cellular cytotoxicity in vitro and in vivo. Based on these data, a clinical trial was performed to test whether interleukin (IL)-2, by increasing FcRgammaIII(+) natural killer (NK) cell numbers and cytolytic function in vivo, when added to trastuzumab, can increase efficacy, be safely given, and avoid the use of chemotherapy. EXPERIMENTAL DESIGN: In this Phase I trial, 10 patients with HER2-overexpressing metastatic breast cancer were treated with IL-2 (1.75 x 10(6) IU/m(2)/day, s.c.) for 7 weeks and trastuzumab (4 mg/kg load and then 2 mg/kg weekly) for 6 weeks. Safety, in vitro immune responses, and clinical responses were assessed. RESULTS: Ten women received a total of 12 cycles of therapy (each cycle lasted 7 weeks). No significant toxicities were seen, and one patient required an IL-2 dose reduction. Among the evaluable patients (10 cycles), the responses were one partial response, five cases of stable disease, and four cases of progressive disease. In vitro immune assays showed NK cell expansion and trastuzumab-mediated increased NK cell killing of breast cancer targets (antibody-dependent cellular cytotoxicity) in a HER2-specific manner but did not correlate with clinical responses. CONCLUSIONS: Trastuzumab + IL-2 is a well-tolerated outpatient regimen that results in NK cell expansion with enhanced in vitro targeted killing of HER2-expressing cells. These preliminary data suggest that this strategy may benefit heavily pretreated metastatic breast cancer patients.