First-Line Venetoclax Combinations in Chronic Lymphocytic Leukemia.

BACKGROUND: Randomized trials of venetoclax plus anti-CD20 antibodies as first-line treatment in fit patients (i.e., those with a low burden of coexisting conditions) with advanced chronic lymphocytic leukemia (CLL) have been lacking. METHODS: In a phase 3, open-label trial, we randomly assigned, in a 1:1:1:1 ratio, fit patients with CLL who did not have TP53 aberrations to receive six cycles of chemoimmunotherapy (fludarabine-cyclophosphamide-rituximab or bendamustine-rituximab) or 12 cycles of venetoclax-rituximab, venetoclax-obinutuzumab, or venetoclax-obinutuzumab-ibrutinib. Ibrutinib was discontinued after two consecutive measurements of undetectable minimal residual disease or could be extended. The primary end points were undetectable minimal residual disease (sensitivity, <10-4 [i.e., <1 CLL cell in 10,000 leukocytes]) as assessed by flow cytometry in peripheral blood at month 15 and progression-free survival. RESULTS: A total of 926 patients were assigned to one of the four treatment regimens (229 to chemoimmunotherapy, 237 to venetoclax-rituximab, 229 to venetoclax-obinutuzumab, and 231 to venetoclax-obinutuzumab-ibrutinib). At month 15, the percentage of patients with undetectable minimal residual disease was significantly higher in the venetoclax-obinutuzumab group (86.5%; 97.5% confidence interval [CI], 80.6 to 91.1) and the venetoclax-obinutuzumab-ibrutinib group (92.2%; 97.5% CI, 87.3 to 95.7) than in the chemoimmunotherapy group (52.0%; 97.5% CI, 44.4 to 59.5; P<0.001 for both comparisons), but it was not significantly higher in the venetoclax-rituximab group (57.0%; 97.5% CI, 49.5 to 64.2; P = 0.32). Three-year progression-free survival was 90.5% in the venetoclax-obinutuzumab-ibrutinib group and 75.5% in the chemoimmunotherapy group (hazard ratio for disease progression or death, 0.32; 97.5% CI, 0.19 to 0.54; P<0.001). Progression-free survival at 3 years was also higher with venetoclax-obinutuzumab (87.7%; hazard ratio for disease progression or death, 0.42; 97.5% CI, 0.26 to 0.68; P<0.001), but not with venetoclax-rituximab (80.8%; hazard ratio, 0.79; 97.5% CI, 0.53 to 1.18; P = 0.18). Grade 3 and grade 4 infections were more common with chemoimmunotherapy (18.5%) and venetoclax-obinutuzumab-ibrutinib (21.2%) than with venetoclax-rituximab (10.5%) or venetoclax-obinutuzumab (13.2%). CONCLUSIONS: Venetoclax-obinutuzumab with or without ibrutinib was superior to chemoimmunotherapy as first-line treatment in fit patients with CLL. (Funded by AbbVie and others; GAIA-CLL13 ClinicalTrials.gov number, NCT02950051; EudraCT number, 2015-004936-36.).

High karyotypic complexity is an independent prognostic factor in patients with CLL treated with venetoclax combinations.

Complex karyotypes have been associated with inferior outcomes in chronic lymphocytic leukemia (CLL) treated with chemoimmunotherapy (CIT), whereas their prognostic impact in the context of venetoclax-based treatments is still debated. In this prospective analysis on karyotype complexity in CLL, we evaluated the impact of complex (≥3 chromosomal aberrations [CAs], CKTs) and highly complex karyotypes (≥5 CAs; hCKTs) as well as specific aberrations in previously untreated patients without TP53 aberrations undergoing either CIT or time-limited venetoclax-based therapies in the phase 3 GAIA/CLL13 trial. Karyotype analyses were available for 895 of 926 patients (96.7%), of whom 153 (17%) had a CKT and 43 (5%) hCKT. In the CIT arm, CKT was associated with shorter progression-free survival (PFS) (hazard ratio [HR] 2.58; 95% confidence interval [95% CI], 1.54-4.32; P < .001) and overall survival (HR, 3.25; 95% CI, 1.03-10.26; P = .044). In the pooled venetoclax arms, a multivariable analysis identified hCKTs (HR, 1.96; 95% CI, 1.03-3.72; P = .041), but not CKTs, as independent adverse prognosticators for PFS. The presence of translocations (unbalanced and/or balanced) was also independently associated with shorter PFSs in the venetoclax arms. CIT led to the acquisition of additional CAs (mean CAs, 2.0-3.4; from baseline to CLL progression), whereas karyotype complexity remained stable after venetoclax-based treatments (2.0, both time points). This analysis establishes highly complex karyotypes and translocations as adverse prognostic factors in the context of venetoclax-based combination treatments. The findings of this study support the incorporation of karyotyping into the standard diagnostic workup of CLL, because it identifies patients at high risk of poor treatment outcomes and thereby improves prognostication. This trial was registered at www.clinicaltrials.gov as #NCT02950051.

First-line venetoclax combinations versus chemoimmunotherapy in fit patients with chronic lymphocytic leukaemia (GAIA/CLL13): 4-year follow-up from a multicentre, open-label, randomised, phase 3 trial.

BACKGROUND: In the primary analysis report of the GAIA/CLL13 trial, we found that venetoclax-obinutuzumab and venetoclax-obinutuzumab-ibrutinib improved undetectable measurable residual disease (MRD) rates and progression-free survival compared with chemoimmunotherapy in patients with previously untreated chronic lymphocytic leukaemia. However, to our knowledge, no data on direct comparisons of different venetoclax-based combinations are available. METHODS: GAIA/CLL13 is an open-label, randomised, phase 3 study conducted at 159 sites in ten countries in Europe and the Middle East. Eligible patients were aged 18 years or older, with a life expectancy of at least 6 months, an Eastern Cooperative Oncology group performance status of 0-2, a cumulative illness rating scale score of 6 or lower or a single score of 4 or lower, and no TP53 aberrations. Patients were randomly assigned (1:1:1:1), with a computer-generated list stratified by age, Binet stage, and regional study group, to either chemoimmunotherapy, venetoclax-rituximab, venetoclax-obinutuzumab, or venetoclax-obinutuzumab-ibrutinib. All treatments were administered in 28-day cycles. Patients in the chemoimmunotherapy group received six cycles of treatment, with patients older than 65 years receiving intravenous bendamustine (90 mg/m2, days 1-2), whereas patients aged 65 years or younger received intravenous fludarabine (25 mg/m2, days 1-3) and intravenous cyclophosphamide (250 mg/m2, days 1-3). Intravenous rituximab (375 mg/m2, day 1 of cycle 1; 500 mg/m2, day 1 of cycles 2-6) was added to chemotherapy. In the experimental groups, patients received daily venetoclax (400 mg orally) for ten cycles after a 5-week ramp-up phase starting on day 22 of cycle 1. In the venetoclax-rituximab group, intravenous rituximab (375 mg/m2, day 1 of cycle 1; 500 mg/m2, day 1 of cycles 2-6) was added. In the obinutuzumab-containing groups, obinutuzumab was added (cycle 1: 100 mg on day 1, 900 mg on day 2, and 1000 mg on days 8 and 15; cycles 2-6: 1000 mg on day 1). In the venetoclax-obinutuzumab-ibrutinib group, daily ibrutinib (420 mg orally, from day 1 of cycle 1) was added until undetectable MRD was reached in two consecutive measurements (3 months apart) or until cycle 36. The planned treatment duration was six cycles in the chemoimmunotherapy group, 12 cycles in the venetoclax-rituximab and the venetoclax-obinutuzumab group and between 12 and 36 cycles in the venetoclax-obinutuzumab-ibrutinib group. Coprimary endpoints were the undetectable MRD rate in peripheral blood at month 15 for the comparison of venetoclax-obinutuzumab versus standard chemoimmunotherapy and investigator-assessed progression-free survival for the comparison of venetoclax-obinutuzumab-ibrutinib versus standard chemoimmunotherapy, both analysed in the intention-to-treat population (ie, all patients randomly assigned to treatment) with a split α of 0·025 for each coprimary endpoint. Both coprimary endpoints have been reported elsewhere. Here we report a post-hoc exploratory analysis of updated progression-free survival results after a 4-year follow-up of our study population. Safety analyses included all patients who received at least one dose of study treatment. This study is registered with ClinicalTrials.gov, NCT02950051, recruitment is complete, and all patients are off study treatment. FINDINGS: Between Dec 13, 2016, and Oct 13, 2019, 1080 patients were screened and 926 were randomly assigned to treatment (chemoimmunotherapy group n=229; venetoclax-rituximab group n=237; venetoclax-obinutuzumab group n=229; and venetoclax-obinutuzumab-ibrutinib group n=231); mean age 60·8 years (SD 10·2), 259 (28%) of 926 patients were female, and 667 (72%) were male (data on race and ethnicity are not reported). At data cutoff for this exploratory follow-up analysis (Jan 31, 2023; median follow-up 50·7 months [IQR 44·6-57·9]), patients in the venetoclax-obinutuzumab group had significantly longer progression-free survival than those in the chemoimmunotherapy group (hazard ratio [HR] 0·47 [97·5% CI 0·32-0·69], p<0·0001) and the venetoclax-rituximab group (0·57 [0·38-0·84], p=0·0011). The venetoclax-obinutuzumab-ibrutinib group also had a significantly longer progression-free survival than the chemoimmunotherapy group (0·30 [0·19-0·47]; p<0·0001) and the venetoclax-rituximab group (0·38 [0·24-0·59]; p<0·0001). There was no difference in progression-free survival between the venetoclax-obinutuzumab-ibrutinib and venetoclax-obinutuzumab groups (0·63 [0·39-1·02]; p=0·031), and the proportional hazards assumption was not met for the comparison between the venetoclax-rituximab group versus the chemoimmunotherapy group (log-rank p=0·10). The estimated 4-year progression-free survival rate was 85·5% (97·5% CI 79·9-91·1; 37 [16%] events) in the venetoclax-obinutuzumab-ibrutinib group, 81·8% (75·8-87·8; 55 [24%] events) in the venetoclax-obinutuzumab group, 70·1% (63·0-77·3; 84 [35%] events) in the venetoclax-rituximab group, and 62·0% (54·4-69·7; 90 [39%] events) in the chemoimmunotherapy group. The most common grade 3 or worse treatment-related adverse event was neutropenia (114 [53%] of 216 patients in the chemoimmunotherapy group, 109 [46%] of 237 in the venetoclax-rituximab group, 127 [56%] of 228 in the venetoclax-obinutuzumab group, and 112 [48%] of 231 in the venetoclax-obinutuzumab-ibrutinib group). Deaths determined to be associated with study treatment by the investigator occurred in three (1%) patients in the chemoimmunotherapy group (n=1 due to each of sepsis, metastatic squamous cell carcinoma, and Richter's syndrome), none in the venetoclax-rituximab and venetoclax-obinutuzumab groups, and four (2%) in the venetoclax-obinutuzumab-ibrutinib group (n=1 due to each of acute myeloid leukaemia, fungal encephalitis, small-cell lung cancer, and toxic leukoencephalopathy). INTERPRETATION: With more than 4 years of follow-up, venetoclax-obinutuzumab and venetoclax-obinutuzumab-ibrutinib significantly extended progression-free survival compared with both chemoimmunotherapy and venetoclax-rituximab in previously untreated, fit patients with chronic lymphocytic leukaemia, thereby supporting their use and further evaluation in this patient group, while still considering the higher toxicities observed with the triple combination. FUNDING: AbbVie, Janssen, and F Hoffmann-La Roche.

Real-world outcomes following third or subsequent lines of therapy: A Danish population-based study on 189 patients with relapsed/refractory large B-cell lymphomas.

Outcome data of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) beyond the second line are scarce outside of clinical trials. Novel therapies in the R/R setting have been approved based on single-arm trials, but results need to be contextualized by real-world outcomes. Medical records from 3753 Danish adults diagnosed with DLBCL were reviewed. Patients previously treated with rituximab and anthracycline-based chemotherapy who received the third or later line (3 L+) of treatment after 1 January 2015, were included. Only 189 patients with a median age of 71 years were eligible. The median time since the last line of therapy was 6 months. Patients were treated with either best supportive care (22%), platinum-based salvage therapy (13%), low-intensity chemotherapy (22%), in clinical trial (14%) or various combination treatments (32%). The 2-year OS-/PFS estimates were 25% and 12% for all patients and 49% and 17% for those treated with platinum-based salvage therapy. Age ≥70, CNS involvement, elevated LDH and ECOG ≥2 predicted poor outcomes, and patients with 0-1 of these risk factors had a 2-year OS estimate of 65%. Only a very small fraction of DLBCL patients received third-line treatment and were eligible for inclusion. Outcomes were generally poor, but better in intensively treated, fit young patients with limited disease.

Survival in multiple myeloma and SARS-COV-2 infection through the COVID-19 pandemic: Results from the EPICOVIDEHA registry.

Patients affected by multiple myeloma (MM) have an increased risk of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection and subsequent coronavirus (20)19 disease (COVID-19)-related death. The changing epidemiological and therapeutic scenarios suggest that there has been an improvement in severity and survival of COVID-19 during the different waves of the pandemic in the general population, but this has not been investigated yet in MM patients. Here we analyzed a large cohort of 1221 patients with MM and confirmed SARS-CoV-2 infection observed between February 2020, and August 2022, in the EPICOVIDEHA registry from 132 centers around the world. Median follow-up was 52 days for the entire cohort and 83 days for survivors. Three-hundred and three patients died (24%) and COVID-19 was the primary reason for death of around 89% of them. Overall survival (OS) was significantly higher in vaccinated patients with both stable and active MM versus unvaccinated, while only a trend favoring vaccinated patients was observed in subjects with responsive MM. Vaccinated patients with at least 2 doses showed a better OS than those with one or no vaccine dose. Overall, according to pandemic waves, mortality rate decreased over time from 34% to 10%. In multivariable analysis, age, renal failure, active disease, hospital, and intensive care unit admission, were independently associated with a higher number of deaths, while a neutrophil count above 0.5 × 109 /L was found to be protective. This data suggests that MM patients remain at risk of SARS-CoV-2 infection even in the vaccination era, but their clinical outcome, in terms of OS, has progressively improved throughout the different viral phases of the pandemic.

COVID-19 severity in patients with chronic lymphocytic leukemia treated with venetoclax: a single-center observational cohort study.

Patients with chronic lymphocytic leukemia (CLL) are at high risk of developing severe COVID-19. The present study was undertaken to elucidate COVID-19 related morbidity and mortality in CLL patients treated with venetoclax. We present a single-center study of 108 patients with small lymphocytic lymphoma or CLL treated with venetoclax. Primary outcome was 30-day COVID-19 mortality. Secondary outcomes included COVID-19 severity and hospitalization rate. Forty-eight (44%) patients had PCR-verified SARS-COV-2 between March 2020 and January 2023. Thirty-six patients (75%) presented with asymptomatic/mild COVID-19 and 12 (25%) with severe/critical disease. The hospitalization rate was 46% with a 30-day mortality rate of only 4% and severe comorbidities as the primary cause of death. COVID-19 severity and mortality were similar before and during the Omicron era. High CIRS-scores (P < 0.02) and thrombocytopenia (P < 0.01) were more frequent in patients with severe/critical disease. In real-world data, most venetoclax treated patients presented with mild COVID-19. Hospitalization and mortality rates were low compared to data of general CLL populations. Our data indicate that venetoclax was a safe treatment option for CLL patients during the pandemic.

Autoimmune blistering disorders and cardiovascular risks: A population-based cohort study.

BACKGROUND: Autoimmune blistering disorders (ABDs) might elevate cardiovascular risk, but studies are lacking. OBJECTIVE: The objective of this study was to examine if ABDs elevate the risk of atherosclerotic cardiovascular disease, heart failure, arrhythmia, venous thromboembolism, and cardiovascular death. METHODS: A population-based cohort of Danish patients with ABD (≥18 years of age) diagnosed during 1996-2021 (n = 3322) was compared with an age- and sex-matched comparison cohort from the general population (n = 33,195). RESULTS: Compared with the general population, patients with ABDs had higher 1-year risks of atherosclerotic cardiovascular disease (3.4% vs 1.6%), heart failure (1.9% vs 0.7%), arrhythmia (3.8% vs 1.3%), venous thromboembolism (1.9% vs 0.3%), and cardiovascular death (3.3% vs 0.9%). The elevated risk persisted after 10 years for all outcomes but arrhythmia. The hazard ratios associating ABDs with the outcomes during the entire follow-up were 1.24 (1.09-1.40) for atherosclerotic cardiovascular disease, 1.48 (1.24-1.77) for heart failure, 1.16 (1.02-1.32) for arrhythmia, 1.87 (1.50-2.34) for venous thromboembolism, and 2.01 (1.76-2.29) for cardiovascular death. The elevated cardiovascular risk was observed for both pemphigus and pemphigoid. LIMITATIONS: Our findings might only generalize to patients with ABDs without prevalent cardiovascular diseases. CONCLUSION: Patients with ABDs had an elevated cardiovascular risk compared with age- and sex-matched controls.

The N-coil and the globular N-terminal domain of plant ARGONAUTE1 are interaction hubs for regulatory factors.

The effector complex of RNA interference (RNAi) contains at its core an ARGONAUTE (AGO) protein bound to a small guide RNA. AGO proteins adopt a two-lobed structure in which the N-terminal (N) and Piwi-Argonaute-Zwille (PAZ) domains make up one lobe, while the middle (MID) and Piwi domains make up the other. Specific biochemical functions of PAZ, MID and Piwi domains of eukaryotic AGO proteins have been described, but the functions of the N domain remain less clear. Here, we use yeast two-hybrid screening with the N domain of the founding member of the AGO protein family, Arabidopsis AGO1, to reveal that it interacts with many factors involved in regulated proteolysis. Interaction with a large group of proteins, including the autophagy cargo receptors ATI1 and ATI2, requires residues in a short, linear region, the N-coil, that joins the MID-Piwi lobe in the three-dimensional structure of AGO. In contrast, the F-box protein AUF1 interacts with AGO1 independently of the N-coil and requires distinct residues in the globular N domain itself. Mutation of AGO1 residues necessary for interaction with protein degradation factors in yeast stabilizes reporters fused to the AGO1 N domain in plants, supporting their in vivo relevance. Our results define distinct regions of the N domain implicated in protein-protein interaction, and point to a particular importance of the AGO1 N-coil as a site of interaction with regulatory factors.

Transesophageal echocardiography of cardiac function in Nile crocodiles - A novel tool for assessing complex hemodynamic patterns.

BACKGROUND: The crocodilian heart is unique among reptiles with its four-chambered structure and complete intracardiac separation of pulmonary and systemic blood flows and pressures. Crocodiles have retained two aortic arches; one from each ventricle, that communicate via Foramen of Panizza, immediately distally from the aortic valves. Moreover, crocodiles can regulate vascular resistance in the pulmonary portion of the right ventricular outflow tract (RVOT). These unique features allow for a complex regulation of shunting between the pulmonary and systemic circulations. Studies on crocodile shunting have predominantly been based on invasive measurements, but here we report on the use of echocardiography. METHODS: Experiments were performed on seven pentobarbital anaesthetized juvenile Nile crocodiles (length and mass of 192 ± 13 cm and 26 ± 5 kg, respectively). Echocardiographic imaging was performed using a transesophageal (TEE) approach. All images were EKG-gated. RESULTS: We obtain excellent views of cardiac structures and central vasculature through the esophagus. Standard imaging planes were defined for both long- and short axis views of the left ventricle and truncus arteriosus. For the RV, only a short axis view could be obtained. Color Doppler was used to visualize flow. Pulsed waved Doppler for measuring flow profiles across the atrioventricular valves, in the two RVOTs and the left ventricular outflow tract. Shunting across the Foramen of Panizza could be visualized and gated to the EKG. CONCLUSION: TEE can be used to image the unique features of the crocodile heart and allow for in-vivo imaging of the complex shunting hemodynamics, including timing of cardiac shunts.

Real-world outcomes upon second-line treatment in patients with chronic lymphocytic leukaemia.

For chronic lymphocytic leukaemia (CLL), targeted drugs have become the standard of care, in particular for second-line treatment. In this study, overall survival (OS), treatment-free survival (TFS) and adverse events (AE) were registered retrospectively in a Danish population-based cohort upon second-line treatment for CLL. Data were collected from medical records and the Danish National CLL register. For 286 patients receiving second-line treatment, three-year TFS was higher upon targeted treatment (ibrutinib/venetoclax/idelalisib) [63%, 95% confidence interval (CI) 50%-76%] compared with fludarabine, cyclophosphamide and rituximab or bendamustine and rituximab (FCR/BR) (37%, CI: 26%-48%) and chlorambucil+/-CD20-antibody (CD20Clb/Clb) (22%, CI: 10%-33%). Upon targeted treatment, three-year OS estimates were higher for targeted treatment (79%, CI: 68%-91%) compared with FCR/BR (70%, CI: 60%-81%) or CD20Clb/Clb (60%, CI: 47%-74%). The most common AEs were infections and haematological AEs; 92% of patients treated with targeted drugs had AEs, 53% of which were severe. Upon FCR/BR and CD20Clb/Clb, AEs were present for 75% and 53% respectively, of which 63% and 31% were severe. These real-world data demonstrate higher TFS and a tendency towards higher OS following targeted second-line treatment for CLL compared to chemoimmunotherapy, also for patients who may be frailer and more comorbid.

NT-proBNP cut-off value for ruling out heart failure in atrial fibrillation patients - A prospective clinical study.

STUDY OBJECTIVE: N-terminal pro-brain natriuretic peptide (NT-proBNP) measurements can be used to rule out heart failure in patients with sinus rhythm. Atrial fibrillation often coexists with heart failure but affects NT-proBNP levels. This study aims to identify the optimal NT-proBNP cut-off value for ruling out heart failure among atrial fibrillation patients. METHODS: This prospective study included 409 atrial fibrillation patients admitted to the emergency department. The inclusion criterion was documented atrial fibrillation on a 12­lead electrocardiogram. All patients completed a NT-proBNP blood sample, a chest X-ray and an echocardiogram. Heart failure was defined as a left ventricular ejection fraction of <40%. RESULTS: In total, 409 patients were included (mean age: 75.2 ± 11.6). The median NT-proBNP level was 2577 ng/L (quartiles: 1185-5438) and 21% had heart failure. We found a lower median NT proBNP level of 3187 ± 3973 ng/L in patients without heart failure compared to 9254 ± 8008 ng/L in patients with heart failure (absolute difference: 4131, 95% (CI): 3299-4986, p < 0.001). The area under the receiver operating characteristic curve for diagnosing heart failure was 0.82 (95% confidence interval: 0.77-0.87). The optimal cut-off value for ruling out heart failure was 739 ng/L with a sensitivity of 99%, a specificity of 18%, and a negative predictive value of 98%. CONCLUSIONS: NT-proBNP can be used to rule out heart failure in atrial fibrillation patients with a high negative predictive value, but low specificity. TRIAL REGISTRATION NUMBER: NCT04125966. https://clinicaltrials.gov/ct2/show/NCT04125966.

Minimal residual disease-guided stop and start of venetoclax plus ibrutinib for patients with relapsed or refractory chronic lymphocytic leukaemia (HOVON141/VISION): primary analysis of an open-label, randomised, phase 2 trial.

BACKGROUND: Targeted time-limited treatment options are needed for patients with relapsed or refractory chronic lymphocytic leukaemia. The aim of this study was to investigate the efficacy of minimal residual disease (MRD)-guided, time-limited ibrutinib plus venetoclax treatment in this patient group. METHODS: HOVON141/VISION was an open-label, randomised, phase 2 trial conducted in 47 hospitals in Belgium, Denmark, Finland, the Netherlands, Norway, and Sweden. Eligible participants were aged 18 years or older with previously treated chronic lymphocytic leukaemia with or without TP53 aberrations; had not been exposed to Bruton tyrosine-kinase inhibitors or BCL2 inhibitors; had a creatinine clearance rate of 30 mL/min or more; and required treatment according to International Workshop on Chronic Lymphocytic Leukemia 2018 criteria. Participants with undetectable MRD (<10-4; less than one chronic lymphocytic leukaemia cell per 10 000 leukocytes) in peripheral blood and bone marrow after 15 28-day cycles of oral ibrutinib (420 mg once daily) plus oral venetoclax (weekly ramp-up 20 mg, 50 mg, 100 mg, 200 mg, up to 400 mg once daily) were randomly assigned (1:2) to ibrutinib maintenance or treatment cessation. Patients who were MRD positive continued to receive ibrutinib monotherapy. Patients who became MRD (>10-2) during observation reinitiated treatment with ibrutinib plus venetoclax. The primary endpoint was progression-free survival at 12 months after random assignment in the treatment cessation group. Progression-free survival was analysed in the intention-to-treat population. All patients who received at least one dose of study drug were included in the safety assessment. The study is registered at ClinicalTrials.gov, NCT03226301, and is active but not recruiting. FINDINGS: Between July 12, 2017, and Jan 21, 2019, 230 patients were enrolled, 225 of whom were eligible. 188 (84%) of 225 completed treatment with ibrutinib plus venetoclax and were tested for MRD at cycle 15. After cycle 15, 78 (35%) patients had undetectable MRD and 72 (32%) were randomly assigned to a treatment group (24 to ibrutinib maintenance and 48 to treatment cessation). The remaining 153 patients were not randomly assigned and continued with ibrutinib monotherapy. Median follow-up of 208 patients still alive and not lost to follow-up at data cutoff on June 22, 2021, was 34·4 months (IQR 30·6-37·9). Progression-free survival after 12 months in the treatment cessation group was 98% (95% CI 89-100). Infections (in 130 [58%] of 225 patients), neutropenia (in 91 [40%] patients), and gastrointestinal adverse events (in 53 [24%] patients) were the most frequently reported; no new safety signals were detected. Serious adverse events were reported in 46 (40%) of 116 patients who were not randomly assigned and who continued ibrutinib maintenance after cycle 15, eight (33%) of 24 patients in the ibrutinib maintenance group, and four (8%) of 48 patients in the treatment cessation group. One patient who was not randomly assigned had a fatal adverse event (bleeding) deemed possibly related to ibrutinib. INTERPRETATION: These data point to a favourable benefit-risk profile of MRD-guided, time-limited treatment with ibrutinib plus venetoclax for patients with relapsed or refractory chronic lymphocytic leukaemia, suggesting that MRD-guided cessation and reinitiation is feasible in this patient population. FUNDING: AbbVie and Janssen.

An m6A-YTH Module Controls Developmental Timing and Morphogenesis in Arabidopsis.

Methylation of N6-adenosine (m6A) in mRNA is an important posttranscriptional gene regulatory mechanism in eukaryotes. m6A provides a binding site for effector proteins ("readers") that influence pre-mRNA splicing, mRNA degradation, or translational efficiency. YT521-B homology (YTH) domain proteins are important m6A readers with established functions in animals. Plants contain more YTH domain proteins than other eukaryotes, but their biological importance remains unknown. Here, we show that the cytoplasmic Arabidopsis thaliana YTH domain proteins EVOLUTIONARILY CONSERVED C-TERMINAL REGION2/3 (ECT2/3) are required for the correct timing of leaf formation and for normal leaf morphology. These functions depend fully on intact m6A binding sites of ECT2 and ECT3, indicating that they function as m6A readers. Mutation of the close ECT2 homolog, ECT4, enhances the delayed leaf emergence and leaf morphology defects of ect2/ect3 mutants, and all three ECT proteins are expressed at leaf formation sites in the shoot apex of young seedlings and in the division zone of developing leaves. ECT2 and ECT3 are also highly expressed at early stages of trichome development and are required for trichome morphology, as previously reported for m6A itself. Overall, our study establishes the relevance of a cytoplasmic m6A-YTH regulatory module in the timing and execution of plant organogenesis.

SARS-CoV-2 infection among patients with haematological disorders: Severity and one-month outcome in 66 Danish patients in a nationwide cohort study.

OBJECTIVES: Patients with haematological disorders may be particularly vulnerable to respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, this is unknown. METHODS: We conducted a prospective, nationwide study including 66 patients in follow-up at Danish haematology departments with a malignant or non-malignant haematological disorder and with verified SARS-CoV-2 infection. Outcomes were intensive care unit (ICU) admission and one-month survival rate. RESULTS: Mean age was 66.7 years, 60.6% were males, 90.9% had comorbidity, and 13.6% had a BMI ≥ 30. The most frequent diagnoses were chronic lymphocytic leukaemia/lymphoma (47.0%), multiple myeloma (16.7%) and acute leukaemia/myelodysplastic syndrome (AL/MDS) (12.1%). Treatment for the haematological disease was ongoing in 59.1% of cases. Neutropenia was present in 6.5%, lymphopenia in 46.6% and hypogammaglobulinaemia in 26.3%. The SARS-CoV-2 infection was mild in 50.0%, severe in 36.4% and critical in 13.6%. After one month, 21.2% had been admitted to ICU, and 24.2% died. Mortality was highest in older patients, patients with severe/critical SARS-CoV-2 infection, high comorbidity score or high performance status score, purine analogue treatment and with AL/MDS. Although older patients and patients with comorbidities had the highest mortality rates, mortality was considerable among all haematological patients. CONCLUSION: Haematological patients with SARS-CoV-2 infection has a severe clinical course.

Rational Development of Stable PYY3-36 Peptide Y2 Receptor Agonists.

PURPOSE: The anorectic effect of PYY3-36 makes it a potential pharmacological weight loss treatment. Modifications of the endogenous peptide to obtain commercially attractive pharmacological and biophysical stability properties are examined. METHODS: Half-life extended PYY3-36 analogues were prepared and examined regarding Y2-receptor potency as well as biophysical and stability properties. RESULTS: Deamidation of asparagine in position 18 and 29 was observed upon incubation at 37°C. Asparagine in position 18 - but not position 29 - could be substituted to glutamine without detrimental effects on Y2-receptor potency. Covalent dimers were formed via the phenol impurity benzoquinone reacting with two N-terminal residues (Isoleucine-Lysine). Both residues had to be modified to suppress dimerization, which could be done without negatively affecting Y2-receptor potency or other stability/biophysical properties. Introduction of half-life extending modifications in position 30 and 35 eliminated aggregation at 37°C without negatively affecting other stability properties. Placement of a protracting moiety (fatty acid) in the receptor-binding C-terminal region reduced Y2-receptor potency substantially, whereas only minor effects of protractor position were observed on structural, biophysical or stability properties. Lipidated PYY3-36 analogues formed oligomers of various sizes depending on primary structure and solution conditions. CONCLUSIONS: By rational design, a chemically and physically stable Y2-receptor selective, half-life extended PYY3-36 peptide has been developed.

Third DWF1 paralog in Solanaceae, sterol Δ24-isomerase, branches withanolide biosynthesis from the general phytosterol pathway.

A large part of chemodiversity of plant triterpenes is due to the modification of their side chains. Reduction or isomerization of double bonds in the side chains is often an important step for the diversification of triterpenes, although the enzymes involved are not fully understood. Withanolides are a large group of structurally diverse C28 steroidal lactones derived from 24-methylenecholesterol. These compounds are found in the Indian medicinal plant Withania somnifera, also known as ashwagandha, and other members of the Solanaceae. The pathway for withanolide biosynthesis is unknown, preventing sustainable production via white biotechnology and downstream pharmaceutical usages. In the present study, based on genome and transcriptome data we have identified a key enzyme in the biosynthesis of withanolides: a DWF1 paralog encoding a sterol Δ24-isomerase (24ISO). 24ISO originated from DWF1 after two subsequent duplication events in Solanoideae plants. Withanolides and 24ISO appear only in the medicinal plants in the Solanoideae, not in crop plants such as potato and tomato, indicating negative selection during domestication. 24ISO is a unique isomerase enzyme evolved from a reductase and as such has maintained the FAD-binding oxidoreductase structure and requirement for NADPH. Using phylogenetic, metabolomic, and gene expression analysis in combination with heterologous expression and virus-induced gene silencing, we showed that 24ISO catalyzes the conversion of 24-methylenecholesterol to 24-methyldesmosterol. We propose that this catalytic step is the committing step in withanolide biosynthesis, opening up elucidation of the whole pathway and future larger-scale sustainable production of withanolides and related compounds with pharmacological properties.

IGHV mutational status and outcome for patients with chronic lymphocytic leukemia upon treatment: a Danish nationwide population-based study.

Patients with chronic lymphocytic leukemia and unmutated immunoglobulin heavy-chain variable region gene (IGHV) have inferior survival from time of treatment in clinical studies. We assessed real-world outcomes based on mutational status and treatment regimen in a nationwide population-based cohort, comprising all 4,135 patients from the Danish chronic lymphocytic leukemia registry diagnosed between 2008 and 2017. In total, 850 patients with known mutational status received treatment: 42% of patients received intensive chemoimmunotherapy consisting of fludarabine, cyclophosphamide plus rituximab, or bendamustine plus rituximab; 27% received chlorambucil in combination with anti-CD20 antibodies or as monotherapy, and 31% received other, less common treatments. No difference in overall survival from time of first treatment according to mutational status was observed, while treatment-free survival from start of first treatment was inferior for patients with unmutated IGHV. The median treatment-free survival was 2.5 years for patients treated with chlorambucil plus anti-CD20, and 1 year for those who received chlorambucil monotherapy. The 3-year treatment-free survival rates for patients treated with fludarabine, cyclophosphamide plus rituximab, and bendamustine plus rituximab were 90% and 91% for those with mutated IGHV, and 76% and 53% for those with unmutated IGHV, respectively, and the 3-year overall survival rates were similar for the two regimens (86-88%). Thus, it appears that, in the real-world setting, patients progressing after intensive chemoimmunotherapy as first-line therapy can be rescued by subsequent treatment, without jeopardizing their long overall survival. Intensive chemoimmunotherapy remains a legitimate option alongside targeted agents, and part of a personalized treatment landscape in chronic lymphocytic leukemia, while improved supportive care and treatment options are warranted for unfit patients.

Prognostic significance of infectious episodes occurring during first-line therapy for diffuse large B-cell lymphoma - A nationwide cohort study.

Infections during first-line therapy for DLBCL are often associated with chemotherapy dose reductions and increased mortality. Systemic infections have also been suggested as beneficial promotors of immunological responses. However, whether there is an association between the timing of an infectious episode and outcome during treatment has not yet been clarified. We investigated how the occurrence and timing of infectious episodes during the first line of treatment for "de novo" DLBCL influenced patient outcome. We used data on DLBCL patients from the Danish Lymphoma Registry, the Danish National Patient Registry, and the Danish National Pathology Registry. Infections were categorized according to type (ICD-10) and time of occurrence after treatment start. "Early" infections were defined as occurring between days 7 and 42 and "late" infections between days 100 and 150 from treatment start. Patients experiencing both "early and late" infections were categorized separately. We used multivariable Cox regression and Kaplan-Meier estimates to assess the association between infections and survival adjusting for NCCN-IPI, sex, comorbidity, and rituximab treatment. We identified 3546 patients, median age 65 years (IQR 56,73). Infectious episodes occurred in 1171 (33%) patients, of which 666 had "early," 303 "late," and 202 both "early and late" events. Patients without registered infections had a 5-year overall survival (OS) rates of 74%. Those with "early," "late," or "early+late" had 5-year OS of 65%, 62%, and 53%, respectively. Compared with patients without any registered infections, hazard rate ratios (HR) were 1.24 (95% CI 1.05-1.47), 1.32 (95% CI 1.06-1.63), and 1.59 (95% CI 1.27-2.00), respectively, in the multivariable model. We observed that infectious episodes during first-line treatment for "de novo" DLBCL occurred in 44% of the patients. Irrespective of timing, patients with infectious episodes had an inferior outcome compared to those without. Outcome patterns were similar for patients registered with sepsis.

The Slicer Activity of ARGONAUTE1 Is Required Specifically for the Phasing, Not Production, of Trans-Acting Short Interfering RNAs in Arabidopsis.

ARGONAUTE1 (AGO1) mediates posttranscriptional silencing by microRNAs (miRNAs) and short interfering RNAS (siRNAs). AGO1-catalyzed RNA cleavage (slicing) represses miRNA targets, but current models also highlight the roles of slicing in formation of siRNAs and siRNA-AGO1 complexes. miRNA-guided slicing is required for biogenesis of phased, trans-acting siRNAs (tasiRNAs), whose cleaved precursor fragments are converted to double-stranded RNA by RNA-dependent RNA polymerase 6 (RDR6). In addition, unwinding of duplex siRNA bound to AGO1 requires passenger strand cleavage in vitro. In this study, we analyze how mutation of four metal ion-coordinating residues of Arabidopsis thaliana AGO1 affects slicer activity in vitro and siRNA function in vivo. We show that while all four residues are required for slicer activity, they do not contribute equally to catalysis. Moreover, passenger strand cleavage is required for assembly of active AGO1-siRNA complexes in vivo, and many AGO1-bound siRNAs are trimmed in the absence of slicer activity. Remarkably, seedlings defective in AGO1 slicer activity produce abundant siRNAs from tasiRNA loci in vivo. These siRNAs depend on RDR6 and SUPPRESSOR OF GENE SILENCING3, but unlike wild-type tasiRNAs, they are unphased. These results demonstrate that slicing is solely required for phase definition of tasiRNAs, and they strongly support recruitment of RDR6 by AGO1 rather than by cleavage fragments.