Long-term treatment with rilzabrutinib in patients with immune thrombocytopenia.

ABSTRACT: Immune thrombocytopenia (ITP) is an autoimmune disease associated with autoantibody-mediated platelet destruction and impaired platelet production, resulting in thrombocytopenia and a predisposition to bleeding. The ongoing, global phase 1/2 study showed that rilzabrutinib, a Bruton tyrosine kinase inhibitor specifically developed to treat autoimmune disorders, could be an efficacious and well-tolerated treatment for ITP. Clinical activity, durability of response, and safety were evaluated in 16 responding patients who continued rilzabrutinib 400 mg twice daily in the long-term extension (LTE) study. At LTE entry, the median platelet count was 87 × 109/L in all patients, 68 × 109/L in those who had rilzabrutinib monotherapy (n = 5), and 156 × 109/L in patients who received concomitant ITP medication (thrombopoietin-receptor agonists and/or corticosteroids, n = 11). At a median duration of treatment of 478 days (range, 303-764), 11 of 16 patients (69%) continued to receive rilzabrutinib. A platelet count of ≥50 × 109/L was reported in 93% of patients for more than half of their monthly visits. The median percentage of LTE weeks with platelet counts ≥30 × 109/L and ≥50 × 109/L was 100% and 88%, respectively. Five patients discontinued concomitant ITP therapy and maintained median platelet counts of 106 × 109/L at 3 to 6 months after stopping concomitant ITP therapy. Adverse events related to treatment were grade 1 or 2 and transient, with no bleeding, thrombotic, or serious adverse events. With continued rilzabrutinib treatment in the LTE, platelet responses were durable and stable over time with no new safety signals. This trial is registered at www.clinicaltrials.gov as #NCT03395210 and www.clinicaltrialsregister.eu as EudraCT 2017-004012-19.

Response to COVID-19 vaccination in patients on cancer therapy: Analysis in a SARS-CoV-2-naïve population.

BACKGROUND: Cancer patients have increased morbidity and mortality from COVID-19, but may respond poorly to vaccination. The Evaluation of COVID-19 Vaccination Efficacy and Rare Events in Solid Tumors (EVEREST) study, comparing seropositivity between cancer patients and healthy controls in a low SARS-CoV-2 community-transmission setting, allows determination of vaccine response with minimal interference from infection. METHODS: Solid tumor patients from The Canberra Hospital, Canberra, Australia, and healthy controls who received COVID-19 vaccination between March 2021 and January 2022 were included. Blood samples were collected at baseline, pre-second vaccine dose and at 1, 3 (primary endpoint), and 6 months post-second dose. SARS-CoV-2 anti-spike-RBD (S-RBD) and anti-nucleocapsid IgG antibodies were measured. RESULTS: Ninety-six solid tumor patients and 20 healthy controls were enrolled, with median age 62 years, and 60% were female. Participants received either AZD1222 (65%) or BNT162b2 (35%) COVID-19 vaccines. Seropositivity 3 months post vaccination was 87% (76/87) in patients and 100% (20/20) in controls (p = .12). Seropositivity was observed in 84% of patients on chemotherapy, 80% on immunotherapy, and 96% on targeted therapy (differences not satistically significant). Seropositivity in cancer patients increased from 40% (6/15) after first dose, to 95% (35/37) 1 month after second dose, then dropped to 87% (76/87) 3 months after second dose. CONCLUSION: Most patients and all controls became seropositive after two vaccine doses. Antibody concentrations and seropositivity showed a decrease between 1 and 3 months post vaccination, highlighting need for booster vaccinations. SARS-CoV-2 infection amplifies S-RBD antibody responses; however, cannot be adequately identified using nucleocapsid serology. This underlines the value of our COVID-naïve population in studying vaccine immunogenicity.

Platelets mediate the clearance of senescent red blood cells by forming prophagocytic platelet-cell complexes.

ABSTRACT: In humans, ∼0.1% to 0.3% of circulating red blood cells (RBCs) are present as platelet-RBC (P-RBC) complexes, and it is 1% to 2% in mice. Excessive P-RBC complexes are found in diseases that compromise RBC health (eg, sickle cell disease and malaria) and contribute to pathogenesis. However, the physiological role of P-RBC complexes in healthy blood is unknown. As a result of damage accumulated over their lifetime, RBCs nearing senescence exhibit physiological and molecular changes akin to those in platelet-binding RBCs in sickle cell disease and malaria. Therefore, we hypothesized that RBCs nearing senescence are targets for platelet binding and P-RBC formation. Confirming this hypothesis, pulse-chase labeling studies in mice revealed an approximately tenfold increase in P-RBC complexes in the most chronologically aged RBC population compared with younger cells. When reintroduced into mice, these complexes were selectively cleared from the bloodstream (in preference to platelet-free RBC) through the reticuloendothelial system and erythrophagocytes in the spleen. As a corollary, patients without a spleen had higher levels of complexes in their bloodstream. When the platelet supply was artificially reduced in mice, fewer RBC complexes were formed, fewer erythrophagocytes were generated, and more senescent RBCs remained in circulation. Similar imbalances in complex levels and senescent RBC burden were observed in humans with immune thrombocytopenia (ITP). These findings indicate that platelets are important for binding and clearing senescent RBCs, and disruptions in platelet count or complex formation and clearance may negatively affect RBC homeostasis and may contribute to the known risk of thrombosis in ITP and after splenectomy.

Rilzabrutinib, an Oral BTK Inhibitor, in Immune Thrombocytopenia.

BACKGROUND: Rilzabrutinib, an oral, reversible covalent inhibitor of Bruton's tyrosine kinase, may increase platelet counts in patients with immune thrombocytopenia by means of dual mechanisms of action: decreased macrophage (Fcγ receptor)-mediated platelet destruction and reduced production of pathogenic autoantibodies. METHODS: In an international, adaptive, open-label, dose-finding, phase 1-2 clinical trial, we evaluated rilzabrutinib therapy in previously treated patients with immune thrombocytopenia. We used intrapatient dose escalation of oral rilzabrutinib over a period of 24 weeks; the lowest starting dose was 200 mg once daily, with higher starting doses of 400 mg once daily, 300 mg twice daily, and 400 mg twice daily. The primary end points were safety and platelet response (defined as at least two consecutive platelet counts of ≥50×103 per cubic millimeter and an increase from baseline of ≥20×103 per cubic millimeter without the use of rescue medication). RESULTS: Sixty patients were enrolled. At baseline, the median platelet count was 15×103 per cubic millimeter, the median duration of disease was 6.3 years, and patients had received a median of four different immune thrombocytopenia therapies previously. All the treatment-related adverse events were of grade 1 or 2 and transient. There were no treatment-related bleeding or thrombotic events of grade 2 or higher. At a median of 167.5 days (range, 4 to 293) of treatment, 24 of 60 patients (40%) overall and 18 of the 45 patients (40%) who had started rilzabrutinib treatment at the highest dose met the primary end point of platelet response. The median time to the first platelet count of at least 50×103 per cubic millimeter was 11.5 days. Among patients with a primary platelet response, the mean percentage of weeks with a platelet count of at least 50×103 per cubic millimeter was 65%. CONCLUSIONS: Rilzabrutinib was active and associated with only low-level toxic effects at all dose levels. The dose of 400 mg twice daily was identified as the dose for further testing. Overall, rilzabrutinib showed a rapid and durable clinical activity that improved with length of treatment. (Funded by Sanofi; ClinicalTrials.gov number, NCT03395210; EudraCT number, 2017-004012-19.).

A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.

R2 and R2* MRI assessment of liver iron content in an undifferentiated diagnostic population with hyperferritinaemia, and impact on clinical decision making.

PURPOSE: To confirm the linear correlation between Ferriscan® R2 (1/T2 Relaxomatry) and R2* (1/T2* Relaxometry) derived 3D Gradient echo (GRE) mDIXON-Quant sequence (Philips) with simultaneous production of a proton density fat fraction (PDFF) in undifferentiated patients with hyperferritinaemia, and to prospectively determine the clinical utility of this tool in these patients by recording the impact on clinical decision-making. MATERIALS AND METHODS: Participants referred to a hospital haematology outpatient clinic for investigation and management of elevated serum ferritin (two serum ferritin levels > 500 µg/L 4 weeks apart) were included in the study. EXCLUSION CRITERIA: contraindications to MRI; clinically relevant investigations for alternative causes of hyperferritinaemia pending; and terminal illness. Thirty-two participants were recruited: 27 men, 5 women. All MRIs performed at 1.5 T. For R2* quantification, 3D six echo GRE sequence (mDIXON-Quant) was acquired. R2 images were acquired over 20 min as dictated and reported by the licensee (Ferriscan®). Clinician interpretation and patient management based on R2* and liver iron content derived from R2 (LICR2) was recorded. Pearson's correlations, linear regression analyses, and ROC curves were calculated. P value <0.05 was considered significant. RESULTS: A high degree of correlation between mean R2* and LICR2 was observed in this novel patient population (slope ±â€¯SE of 43.35 ±â€¯1.88 s-1 permg/g; 95 % CI 39.5-47.2; P < 0.001; R2 = 0.87). Clinical decision making was amended in 14/32 (44 %) patients with hyperferritinaemia following the disclosure of R2* results to clinicians, compared with serum ferritin alone. Liver biopsy was avoided in one patient based on LICR2 and R2*. Unrecognised hepatic steatosis was diagnosed in one patient from the PDFF map. CONCLUSION: We have confirmed the linear correlation between R2 and R2* in a real-world diagnostic population with hyperferritinaemia. Non-invasive assessment of liver iron content (LIC) by R2 and R2* MRI is a useful clinical tool and alters management in these patients.

The molecular basis of immune-based platelet disorders.

Platelets have a predominant role in haemostasis, the maintenance of blood volume and emerging roles as innate immune cells, in wound healing and in inflammatory responses. Platelets express receptors that are important for platelet adhesion, aggregation, participation in inflammatory responses, and for triggering degranulation and enhancing thrombin generation. They carry a cargo of granules bearing enzymes, adhesion molecules, growth factors and cytokines, and have the ability to generate reactive oxygen species. The platelet is at the frontline of a host of cellular responses to invading pathogens, injury, and infection. Perhaps because of this intrinsic responsibility of a platelet to rapidly respond to thrombotic, pathological and immunological factors as part of their infantry role; platelets are susceptible to targeted attack by the adaptive immune system. Such attacks are often transitory but result in aberrant platelet activation as well as significant loss of platelet numbers and platelet function, paradoxically leading to elevated risks of both thrombosis and bleeding. Here, we discuss the main molecular events underlying immune-based platelet disorders with specific focus on events occurring at the platelet surface leading to activation and clearance.

Novel scientific approaches and future research directions in understanding ITP.

Diagnosis of immune thrombocytopenia (ITP) and prediction of response to therapy remain significant and constant challenges in hematology. In patients who present with ITP, the platelet count is frequently used as a surrogate marker for disease severity, and so often determines the need for therapy. Although there is a clear link between thrombocytopenia and hemostasis, a direct correlation between the extent of thrombocytopenia and bleeding symptoms, especially at lower platelet counts is lacking. Thus, bleeding in ITP is heterogeneous, unpredictable, and nearly always based on a multitude of risk factors, beyond the platelet count. The development of an evidence-based, validated risk stratification model for ITP treatment is a major goal in the ITP community and this review discusses new laboratory approaches to evaluate the various pathobiologies of ITP that may inform such a model.

Immune thrombocytopenia: antiplatelet autoantibodies inhibit proplatelet formation by megakaryocytes and impair platelet production in vitro.

Primary immune thrombocytopenia is an autoimmune disease mediated by antiplatelet autoantibodies that cause platelet destruction and suppression of platelet production. In vitro effects of autoantibodies on megakaryocyte production and maturation have been reported recently. However, the impact of these autoantibodies on crucial megakaryocyte functions, proplatelet formation and subsequent platelet release, has not been evaluated. We examined the effects of serum and IgG from 19 patients with immune thrombocytopenia using day 8 or 9 megakaryocytes (66.3 ± 10.6% CD41(+)), derived from cord blood hematopoietic stem cells (CD34(+)). The number of proplatelet-bearing megakaryocytes, the number of platelets released in the culture, total megakaryocyte numbers, ploidy pattern and caspase activation were measured at various times after treatment. After 5 days of treatment the number of proplatelet-bearing megakaryocytes was significantly decreased by 13 immune thrombocytopenia autoantibodies relative to the control group (P<0.0001) and this decrease was accompanied by a corresponding reduction of platelet release. Other features, including total megakaryocyte numbers, maturation and apoptosis, were not affected by immune thrombocytopenia antibodies. Treating the megakaryocytes with the thrombopoietin receptor agonists romiplostim and eltrombopag reversed the effect of the autoantibodies on megakaryocytes by restoring their capacity to form proplatelets. We conclude that antiplatelet antibodies in immune thrombocytopenia inhibit proplatelet formation by megakaryocytes and hence the ability of the megakaryocytes to release platelets. Treatment with either romiplostim or eltrombopag regenerates proplatelet formation from the megakaryocytes.