Ropeginterferon phase 2 randomized study in low-risk polycythemia vera: 5-year drug survival and efficacy outcomes.

In patients with low-risk polycythemia vera, exposure to low-dose Ropeginterferon alfa-2b (Ropeg) 100 µg every 2 weeks for 2 years was more effective than the standard treatment of therapeutic phlebotomy in maintaining target hematocrit (HCT) (< 45%) with a reduction in the need for phlebotomy without disease progression. In the present paper, we analyzed drug survival, defined as a surrogate measure of the efficacy, safety, adherence, and tolerability of Ropeg in patients followed up to 5 years. During the first 2 years, Ropeg and phlebotomy-only (Phl-O) were discontinued in 33% and 70% of patients, respectively, for lack of response (12 in the Ropeg arm vs. 34 in the Phl-O arm) or adverse events (6 vs. 0) and withdrawal of consent in (3 vs. 10). Thirty-six Ropeg responders continued the drug for up to 3 years, and the probability of drug survival after a median of 3.15 years was 59%. Notably, the primary composite endpoint was maintained in 97%, 94%, and 94% of patients still on drug at 3, 4, and 5 years, respectively, and 60% of cases were phlebotomy-free. Twenty-three of 63 Phl-O patients (37%) failed the primary endpoint and were crossed over to Ropeg; among the risk factors for this failure, the need for more than three bloodletting procedures in the first 6 months emerged as the most important determinant. In conclusion, to improve the effectiveness of Ropeg, we suggest increasing the dose and using it earlier driven by high phlebotomy need in the first 6 months post-diagnosis.

Evans syndrome: Disease awareness and clinical management in a nation-wide ITP-NET survey.

Evans syndrome (ES) is rare and mostly treated on a "case-by-case" basis and no guidelines are available. With the aim of assessing disease awareness and current management of adult ES, a structured survey was administered to 64 clinicians from 50 Italian participating centers. Clinicians had to be involved in the management of autoimmune cytopenias and were enrolled into the ITP-NET initiative. The survey included domains on epidemiology, diagnosis, and therapy of ES and was designed to capture current practice and suggested work-up and management. Thirty clinicians who had followed a median of 5 patients (1-45)/15 years responded. The combination of AIHA plus ITP was more common than the ITP/AIHA with neutropenia (p < .001) and 25% of patients had an associated condition, including lymphoproliferative syndromes, autoimmune diseases, or primary immunodeficiencies. The agreement of clinicians for each diagnostic test is depicted (i.e., 100% for blood count and DAT; only 40% for anti-platelets and anti-neutrophils; 77% for bone marrow evaluation). Most clinicians reported that ES requires a specific approach compared to isolated autoimmune cytopenias, due to either a more complex pathogenesis and a higher risk of relapse and thrombotic and infectious complications. The heterogeneity of treatment choices among different physicians suggests the need for broader harmonization.

CALR exon 9 mutations are somatically acquired events in familial cases of essential thrombocythemia or primary myelofibrosis.

Somatic mutations in the calreticulin (CALR) gene were recently discovered in patients with sporadic essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations. We studied CALR mutation status in familial cases of myeloproliferative neoplasm. In a cohort of 127 patients, CALR indels were identified in 6 of 55 (11%) subjects with ET and in 6 of 20 (30%) with PMF, whereas 52 cases of polycythemia vera had nonmutated CALR. All CALR mutations were somatic, found in granulocytes but not in T lymphocytes. Patients with CALR-mutated ET showed a higher platelet count (P = .017) and a lower cumulative incidence of thrombosis (P = .036) and of disease progression (P = .047) compared with those with JAK2 (V617F). In conclusion, a significant proportion of familial ET and PMF nonmutated for JAK2 carry a somatic mutation of CALR.

JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes.

Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials.

Ropeginterferon versus Standard Therapy for Low-Risk Patients with Polycythemia Vera.

BACKGROUND: Whether phlebotomy alone can adequately maintain target hematocrit in patients with low-risk polycythemia vera (PV) remains elusive. METHODS: In a phase 2 open-label randomized trial, we compared ropeginterferon alfa-2b (ropeg; 100 µg every 2 weeks) with phlebotomy only regarding maintenance of a median hematocrit level (≤45%) over 12 months in the absence of progressive disease (primary end point). In follow-up, crossover to the alternative treatment group was allowed if the primary end point was not met. RESULTS: In total, 127 patients were enrolled (ropeg: n=64; standard group: n=63). The primary end point was met in 81% and 51% in the ropeg and standard groups, respectively. Responders continued the assigned treatment until month 24 and maintained response in 83% and 59%, respectively (P=0.02). Ropeg responders less frequently experienced moderate/severe symptoms (33% vs. 67% in the standard group) and palpable splenomegaly (14% vs. 37%) and showed normalization of ferritin levels and blood counts. Nonresponders at 12 months crossed over to the standard (n=9) or ropeg (n=23) group; in patients switched to ropeg only, 7 of 23 met the response criteria in 12 months, and phlebotomy need was high (4.7 per patient per year). Discontinuation because of adverse events occurred in seven patients treated with ropeg. CONCLUSIONS: In this 24-month trial, ropeg was superior to phlebotomy alone in maintaining hematocrit on target. No dose-limiting side effects or toxicities were noted; 9.2% of patients on ropeg and no patients on standard treatment developed neutropenia. (Funded by AOP Health and others; ClinicalTrials.gov number, NCT03003325.)

The MDM2 antagonist idasanutlin in patients with polycythemia vera: results from a single-arm phase 2 study.

Idasanutlin, an MDM2 antagonist, showed clinical activity and a rapid reduction in JAK2 V617F allele burden in patients with polycythemia vera (PV) in a phase 1 study. This open-label phase 2 study evaluated idasanutlin in patients with hydroxyurea (HU)-resistant/-intolerant PV, per the European LeukemiaNet criteria, and phlebotomy dependence; prior ruxolitinib exposure was permitted. Idasanutlin was administered once daily on days 1 through 5 of each 28-day cycle. The primary end point was composite response (hematocrit control and spleen volume reduction > 35%) in patients with splenomegaly and hematocrit control in patients without splenomegaly at week 32. Key secondary end points included safety, complete hematologic response (CHR), patient-reported outcomes, and molecular responses. All patients (n = 27) received idasanutlin; 16 had response assessment (week 32). Among responders with baseline splenomegaly (n = 13), 9 (69%) attained any spleen volume reduction, and 1 achieved composite response. Nine patients (56%) achieved hematocrit control, and 8 patients (50%) achieved CHR. Overall, 43% of evaluable patients (6/14) showed a ≥50% reduction in the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score (week 32). Nausea (93%), diarrhea (78%), and vomiting (41%) were the most common adverse events, with grade ≥ 3 nausea or vomiting experienced by 3 patients (11%) and 1 patient (4%), respectively. Reduced JAK2 V617F allele burden occurred early (after 3 cycles), with a median reduction of 76%, and was associated with achieving CHR and hematocrit control. Overall, the idasanutlin dosing regimen showed clinical activity and rapidly reduced JAK2 allele burden in patients with HU-resistant/- intolerant PV but was associated with low-grade gastrointestinal toxicity, leading to poor long-term tolerability. This trial was registered at www.clinincaltrials.gov as #NCT03287245.

Universal Newborn Screening for Congenital Cytomegalovirus Infection - From Infant to Maternal Infection: A Prospective Multicenter Study.

Introduction: Most infants at risk for cytomegalovirus (CMV)-associated sensorineural hearing loss (SNHL) are unrecognized because of the absence of a universal neonatal CMV screening. The search of CMV-DNA by molecular methods in salivary swabs was demonstrated to be a reliable approach. This study describes the results obtained by carrying out a universal screening for congenital CMV (cCMV) infection including all live-born newborns in three Italian sites, as well as the therapeutic interventions and clinical outcome of the CMV-infected neonates. Moreover, CMV maternal infection's characteristics were evaluated. Methods: To confirm or exclude cCMV infection, a CMV-DNA-positive result on a first salivary swab was followed by repeated saliva and urine samples collected within 21 days of age. Breast milk samples were also collected. The search of CMV-DNA was performed with a single automated quantitative commercial real-time PCR assay, regardless of the type of samples used. Results: A total of 3,151 newborns were enrolled; 21 (0.66%) of them were congenitally infected (median saliva viral load at screening, 6.65 [range, 5.03-7.17] log10 IU/ml). Very low/low viral load in screening saliva samples (median value, 1.87 [range, 1.14-2.59] log10 IU/ml) was associated with false-positive results (n = 54; 1.7%). CMV-DNA was detected in almost half of the breast milk samples of mother-infant pairs with a false-positive result, suggesting that contamination from breast milk may not be the only explanation in the study population. cCMV infection confirmation with the search of CMV-DNA in a urine sample proved to be the gold standard strategy, since false-positive results were observed in 4/54 (7.5%) of the repeated saliva samples. Symptomatic cCMV infection was observed in 3/21 (14.3%) infants; notably, one (4.7%) developed moderate unilateral SNHL at 5 months after birth. Finally, two symptomatic cCMV infections were associated with primary maternal infection acquired in the first trimester of gestation; one newborn with severe cCMV symptoms was born to a mother with no CMV checkups in pregnancy. Conclusion: Without universal neonatal CMV screening, some infected infants who develop late neurological sequelae may not be recognized and, consequently, they are not able to benefit early from instrumental and therapeutic interventions to limit and/or treat CMV disease.

Ropeginterferon alfa-2b versus phlebotomy in low-risk patients with polycythaemia vera (Low-PV study): a multicentre, randomised phase 2 trial.

BACKGROUND: There is no evidence that phlebotomy alone is sufficient to steadily maintain haematocrit on target level in low-risk patients with polycythaemia vera. This study aimed to compare the efficacy and safety of ropeginterferon alfa-2b on top of the standard phlebotomy regimen with phlebotomy alone. METHODS: In 2017, we launched the Low-PV study, a multicentre, open-label, two-arm, parallel-group, investigator-initiated, phase 2 randomised trial with a group-sequential adaptive design. The study involved 21 haematological centres across Italy. Participants were recruited in a consecutive order. Participants enrolled in the study were patients, aged 18-60 years, with a diagnosis of polycythaemia vera according to 2008-16 WHO criteria. Eligible patients were randomly allocated (1:1) to receive either phlebotomy and low-dose aspirin (standard group) or ropeginterferon alfa-2b on top of the standard treatment (experimental group). Randomisation sequence was generated using five blocks of variable sizes proportional to elements of Pascal's triangle. Allocation was stratified by age and time from diagnosis. No masking was done. Patients randomly allocated to the standard group were treated with phlebotomy (300 mL for each phlebotomy to maintain the haematocrit values of lower than 45%) and low-dose aspirin (100 mg daily), if not contraindicated. Patients randomly allocated to the experimental group received ropeginterferon alfa-2b subcutaneously every 2 weeks in a fixed dose of 100 µg on top of the phlebotomy-only regimen. The primary endpoint was treatment response, defined as maintenance of the median haematocrit values of 45% or lower without progressive disease during a 12-month period. Analyses were done by intention-to-treat principle. The study was powered assuming a higher percentage of responders in the experimental group (75%) than in the standard group (50%). Here we report results from the second planned interim analysis when 50 patients had been recruited to each group. The trial is ongoing, and registered with ClinicalTrials.gov, NCT03003325. FINDINGS: Between Feb 2, 2017, and March 13, 2020, 146 patients were screened, and 127 patients were randomly assigned to the standard group (n=63) or the experimental group (n=64). The median follow-up period was 12·1 months (IQR 12·0-12·6). For the second pre-planned interim analysis, a higher response rate in the experimental group was seen (42 [84%] of 50 patients) than in the standard group (30 [60%] of 50 patients; absolute difference 24%, 95% CI 7-41%, p=0·0075). The observed z value (2·6001) crossed the critical bound of efficacy (2·5262), and the stagewise adjusted p value early showed superiority of experimental treatment. Thus, the data safety monitoring board decided to stop patient accrual for overwhelming efficacy and to continue the follow-up, as per protocol, for 2 years. Under the safety profile, no statistically significant difference between groups in frequency of adverse events of grade 3 or higher was observed; the most frequently reported adverse events were neutropenia (four [8%] of 50 patients) in the experimental group and skin symptoms (two [4%] of 50 patients) in the standard group. No grade 4 or 5 adverse events occurred. INTERPRETATION: Supplementing phlebotomy with ropeginterferon alfa-2b seems to be safe and effective in steadily maintaining haematocrit values on target in low-risk patients with polycythaemia vera. Findings from the current study might have implications for changing the current management of low-risk patients with polycythaemia vera. FUNDING: AOP Orphan Pharmaceuticals, Associazione Italiana per la Ricerca sul Cancro.

Among classic myeloproliferative neoplasms, essential thrombocythemia is associated with the greatest risk of venous thromboembolism during COVID-19.

In a multicenter European retrospective study including 162 patients with COVID-19 occurring in essential thrombocythemia (ET, n = 48), polycythemia vera (PV, n = 42), myelofibrosis (MF, n = 56), and prefibrotic myelofibrosis (pre-PMF, n = 16), 15 major thromboses (3 arterial and 12 venous) were registered in 14 patients, of whom all, but one, were receiving LMW-heparin prophylaxis. After adjustment for the competing risk of death, the cumulative incidence of arterial and venous thromboembolic events (VTE) reached 8.5% after 60 days follow-up. Of note, 8 of 12 VTE were seen in ET. Interestingly, at COVID-19 diagnosis, MPN patients had significantly lower platelet count (p < 0.0001) than in the pre-COVID last follow-up.This decline was remarkably higher in ET (-23.3%, p < 0.0001) than in PV (-16.4%, p = 0.1730) and was associated with higher mortality rate (p = 0.0010) for pneumonia. The effects of possible predictors of thrombosis, selected from those clinically relevant and statistically significant in univariate analysis, were examined in a multivariate model. Independent risk factors were transfer to ICU (SHR = 3.73, p = 0.029), neutrophil/lymphocyte ratio (SHR = 1.1, p = 0.001) and ET phenotype (SHR = 4.37, p = 0.006). The enhanced susceptibility to ET-associated VTE and the associated higher mortality for pneumonia may recognize a common biological plausibility and deserve to be delved to tailor new antithrombotic regimens including antiplatelet drugs.

High mortality rate in COVID-19 patients with myeloproliferative neoplasms after abrupt withdrawal of ruxolitinib.

We report the clinical presentation and risk factors for survival in 175 patients with myeloproliferative neoplasms (MPN) and COVID-19, diagnosed between February and June 2020. After a median follow-up of 50 days, mortality was higher than in the general population and reached 48% in myelofibrosis (MF). Univariate analysis, showed a significant relationship between death and age, male gender, decreased lymphocyte counts, need for respiratory support, comorbidities and diagnosis of MF, while no association with essential thrombocythemia (ET), polycythemia vera (PV), and prefibrotic-PMF (pre-PMF) was found. Regarding MPN-directed therapy ongoing at the time of COVID-19 diagnosis, Ruxolitinib (Ruxo) was significantly more frequent in patients who died in comparison with survivors (p = 0.006). Conversely, multivariable analysis found no effect of Ruxo alone on mortality, but highlighted an increased risk of death in the 11 out of 45 patients who discontinued treatment. These findings were also confirmed in a propensity score matching analysis. In conclusion, we found a high risk of mortality during COVID-19 infection among MPN patients, especially in MF patients and/or discontinuing Ruxo at COVID-19 diagnosis. These findings call for deeper investigation on the role of Ruxo treatment and its interruption, in affecting mortality in MPN patients with COVID-19.

Repurposing of Plasminogen: An Orphan Medicinal Product Suitable for SARS-CoV-2 Inhalable Therapeutics.

The SARS-CoV-2 infection is associated with pulmonary coagulopathy, which determines the deposition of fibrin in the air spaces and lung parenchyma. The resulting lung lesions compromise patient pulmonary function and increase mortality, or end in permanent lung damage for those who have recovered from the COVID-19 disease. Therefore, local pulmonary fibrinolysis can be efficacious in degrading pre-existing fibrin clots and reducing the conversion of lung lesions into lasting scars. Plasminogen is considered a key player in fibrinolysis processes, and in view of a bench-to-bedside translation, we focused on the aerosolization of an orphan medicinal product (OMP) for ligneous conjunctivitis: human plasminogen (PLG-OMP) eye drops. As such, the sterile and preservative-free solution guarantees the pharmaceutical quality of GMP production and meets the Ph. Eur. requirements of liquid preparations for nebulization. PLG-OMP aerosolization was evaluated both from technological and stability viewpoints, after being submitted to either jet or ultrasonic nebulization. Jet nebulization resulted in a more efficient delivery of an aerosol suitable for pulmonary deposition. The biochemical investigation highlighted substantial protein integrity maintenance with the percentage of native plasminogen band > 90%, in accordance with the quality specifications of PLG-OMP. In a coherent way, the specific activity of plasminogen is maintained within the range 4.8-5.6 IU/mg (PLG-OMP pre-nebulization: 5.0 IU/mg). This is the first study that focuses on the technological and biochemical aspects of aerosolized plasminogen, which could affect both treatment efficacy and clinical dosage delivery. Increasing evidence for the need of local fibrinolytic therapy could merge with the availability of PLG-OMP as an easy handling solution, readily aerosolizable for a fast translation into an extended clinical efficacy assessment in COVID-19 patients.

Straightforward recursive partitioning model for discarding insoluble compounds in the drug discovery process.

Poor aqueous solubility is one of the major issues in drug discovery and development, impacting negatively on all aspects of the research and development process. The pharmaceutical industry has realized that solubility issues need to be resolved at the discovery stage. We here present an innovative way to address this problem via a model designed to address the simple question, "Is the compound likely to be sufficiently soluble to provide interpretable data in biological screening assays?" A recursive partitioning (RP) method was applied to a set of 3563 molecules, with in house determined aqueous solubility values. Five models were generated on the basis of a small number of descriptors affording intuitive information regarding structural features influencing solubility. The final model was based on only two descriptors: the molecular weight (MW) and the aromatic proportion (AP). This model provided satisfactory values of accuracy (81%) and precision (75%) for a test set of 1200 compounds, suggesting that the model may add value in compound selection and library design during early drug discovery.

Benefit-risk profile of cytoreductive drugs along with antiplatelet and antithrombotic therapy after transient ischemic attack or ischemic stroke in myeloproliferative neoplasms.

We analyzed 597 patients with myeloproliferative neoplasms (MPN) who presented transient ischemic attacks (TIA, n = 270) or ischemic stroke (IS, n = 327). Treatment included aspirin, oral anticoagulants, and cytoreductive drugs. The composite incidence of recurrent TIA and IS, acute myocardial infarction (AMI), and cardiovascular (CV) death was 4.21 and 19.2%, respectively at one and five years after the index event, an estimate unexpectedly lower than reported in the general population. Patients tended to replicate the first clinical manifestation (hazard ratio, HR: 2.41 and 4.41 for recurrent TIA and IS, respectively); additional factors for recurrent TIA were previous TIA (HR: 3.40) and microvascular disturbances (HR: 2.30); for recurrent IS arterial hypertension (HR: 4.24) and IS occurrence after MPN diagnosis (HR: 4.47). CV mortality was predicted by age over 60 years (HR: 3.98), an index IS (HR: 3.61), and the occurrence of index events after MPN diagnosis (HR: 2.62). Cytoreductive therapy was a strong protective factor (HR: 0.24). The rate of major bleeding was similar to the general population (0.90 per 100 patient-years). In conclusion, the long-term clinical outcome after TIA and IS in MPN appears even more favorable than in the general population, suggesting an advantageous benefit-risk profile of antithrombotic and cytoreductive treatment.

Sequential evaluation of CALR mutant burden in patients with myeloproliferative neoplasms.

We investigated the variation of CALR-mutant burden during follow-up in 105 CALR-mutant MPN and compared it to the variation of JAK2-mutant burden in 226 JAK2-mutant MPN.The median allele burden at last evaluation was significantly higher than at first evaluation in essential thrombocythemia (ET) (49.5% vs 45%, P < .001) but not in primary myelofibrosis (PMF) (52% vs 51%, P 0.398). Median values of slope were positive both in ET (0.071) and in PMF (0.032). In CALR-mutant ET there was a difference between natural and therapy-related slope (P 0.006).In the JAK2-mutated cohort, the median allele burden at last evaluation was not different respect to that at first evaluation, neither in ET (22.9% vs 23.2%, P = 0.216) nor in PMF (50.5% vs 45.0%, P = 0.809), despite a positive slope. Multivariate analysis to evaluate the effect of mutation (CALR vs JAK2) on the slope of mutant burden in not treated pts with a positive slope adjusting for diagnosis (ET vs PMF) showed a trend toward a higher increase of mutant burden in CALR vs JAK2 (ß = 0.19, P = 0.061) with no difference between diagnosis (P = 0.419). The findings of this study suggest that clonal expansion in CALR-mutant MPN is faster than that observed in JAK2-mutant MPN.