The R2 (lenalidomide-rituximab) and R2 with bendamustine (R2B) regimens are both feasible in relapsed/refractory follicular lymphoma (R/R FL), but prospective phase II data on R2B are lacking. In this multinational, prospective, randomised, non-comparative trial, patients with R/R FL were randomised 1:1 to R2 (arm A) or R2B (arm B). Patients achieving CT-based partial or complete remission (PR/CR) received rituximab maintenance every 3 months for 2 years. Co-primary endpoints were investigator-assessed CR rates at end-of-induction (EOI) and severe toxicity rates. Between December 2014 and July 2019, 92 patients were randomized. The trial was stopped prematurely due to slow accrual. CR rates at EOI were 11.4% (95% CI [3.8, 24.6%]) for R2 and 15.2% (CI [6.3, 28.9%]) for R2B. With a median follow-up of 74 months, predefined severe toxicities occurred in 3 (6.8%; CI [1.4, 18.7%]) and 6 pts (13.0%, CI [4.9, 26.3%]) in Arm A and Arm B respectively, with 2 pneumonia-related deaths in arm A and no treatment-related deaths in arm B; 43% and 66% experienced any grade 3-4 AE (2%/7% grade 4). At 60 months, in arms A and B event free survival (EFS) was 39.5% (CI [25.1, 53.6%]) and 56.4% (CI [40.1, 69.3%]). Overall survival (OS) was excellent at 72.1% and 86.3%, respectively. R2 and R2B are both effective treatment regimens for R/R FL. While CT-based CR rates were low, treatment with R2B resulted in numerically longer median EFS and OS, but this was associated with higher toxicity.

Curative-intent immunochemotherapy fails in ∼30% of patients with large B cell lymphoma (LBCL), yet no validated molecular tool enables early identification of high-risk individuals to guide treatment intensification. Using shallow whole-genome sequencing (sWGS) of plasma cell-free DNA from 190 LBCL patients, we develop and validate the ACT score (aberrations, composition of fragments, and terminal motif analyses), a composite classifier integrating genomic and fragmentomic features from a single post-cycle-1 sample. ACT-positive patients have worse 2-year outcomes versus ACT-negative patients: time-to-progression 29% vs. 83% (hazard ratio [HR]: 4.4, 95% confidence interval [CI]: 1.9-10.0; p = 1.5 × 10-4) and overall survival 47% vs. 93% (HR: 8.7, 95% CI: 3.0-25.4; p = 1.8 × 10-6). The ACT score is independently prognostic of the International Prognostic Index, and their combination identifies the highest risk patients. Unlike mutation-based approaches, this assay requires neither tumor tissue, germline control, nor a baseline plasma sample. Built on open-source tools and sWGS, the ACT score offers a feasible, scalable strategy for early risk stratification in aggressive LBCL.

Abstract:Thrombocytopenia at admission predicts mortality in multiple myeloma (MM) and might link to disease progression. Although thrombocytopenia is known to be associated with MM, a possible thrombopathy is clinically less known. We conducted a case-control study comparing platelet responses of MM patients to controls via flow cytometry, integrin αIIbβ3 activation and P-selectin exposure, and a bioluminescent assay, ATP release. No difference was found at baseline, but upon platelet stimulation, MM patients had decreased αIIbβ3 activation, partly impaired P-selectin exposure, and reduced δ-granule (ATP) secretion. Aspirin treatment in patients did not account for these diminished platelet responses. In total, 29% of patients had thrombocytopenia, while 60% had decreased αIIbβ3 activation and 67% had reduced platelet secretion capacity. Importantly, as secretion capacity was corrected for platelet count, granule release per platelet was reduced in patients versus controls. Of 6 patients with thrombocytopenia 4 displayed a thrombopathy, while for 15 patients with normal count, 64% had reduced αIIbβ3 activation and 73% had reduced platelet secretion capacity. Of all patients, 10% had thrombocytopenia combined with reduced αIIbβ3 activation plus low secretion capacity (one patient showed no qualitative or quantitative platelet defect). Our data suggest that beyond the known thrombocytopenia, MM patients also have reduced platelet function, which could reflect impaired platelet vitality. Combined measurement of platelet count and function, especially secretion capacity, gives a more comprehensive view of platelet phenotype than count alone. Large prospective follow-up studies are needed to confirm the importance of the acquired platelet secretion defect on the prognosis of MM patients.

Eosinophilia is a common laboratory finding with a broad differential diagnosis. Eosinophilia can be classified as primary, secondary, or idiopathic hypereosinophilic syndrome (HES). Primary eosinophilia results from a clonal disorder of eosinophils, while secondary eosinophilia is reactive to a variety of underlying conditions, such as allergic diseases, infections, and drug reactions. Idiopathic HES is a diagnosis of exclusion in patients with persistent hypereosinophilia and end-organ damage without an identifiable cause. The diagnostic workup should focus on identifying the cause and assessing for end-organ damage. The initial evaluation should include a detailed history and physical examination, and laboratory tests to assess for organ involvement. Further testing may be necessary based on the clinical presentation and laboratory findings. The treatment depends on the cause. In cases of secondary eosinophilia, treatment should be directed at the underlying condition.

Introduction The risk of major bleeding complications in catheter directed thrombolysis (CDT) for acute limb ischemia (ALI) remains high, with reported major bleeding complication rates in up to 1 in every 10 treated patients. Fibrinogen was the only predictive marker used for bleeding complications in CDT, despite the lack of high quality evidence to support this. Therefore, recent international guidelines recommend against the use of fibrinogen during CDT. However, no alternative biomarkers exist to effectively predict CDT-related bleeding complications. The aim of the POCHET biobank is to prospectively assess the rate and etiology of bleeding complications during CDT and to provide a biobank of blood samples to investigate potential novel biomarkers to predict bleeding complications during CDT. Methods The POCHET biobank is a multicentre prospective biobank. After informed consent, all consecutive patients with lower extremity ALI eligible for CDT are included. All patients are treated according to a predefined standard operating procedure which is aligned in all participating centres. Baseline and follow-up data are collected. Prior to CDT and subsequently every six hours, venous blood samples are obtained and stored in the biobank for future analyses. The primary outcome is the occurrence of non-access related major bleeding complications, which is assessed by an independent adjudication committee. Secondary outcomes are non-major bleeding complications and other CDT related complications. Proposed biomarkers to be investigated include fibrinogen, to end the debate on its usefulness, anti-plasmin and D-Dimer. Discussion and conclusion The POCHET biobank provides contemporary data and outcomes of patients during CDT for ALI, coupled with their blood samples taken prior and during CDT. Thereby, the POCHET biobank is a real world monitor on biomarkers during CDT, supporting a broad spectrum of future research for the identification of patients at high risk for bleeding complications during CDT and to identify new biomarkers to enhance safety in CDT treatment.

Background The MYC oncogene exerts numerous tumor-promoting functions and may enable anti-tumor immune escape by contributing to the remodeling of the tumor microenvironment (TME, PMID 33081056). In light of the development of novel immunotherapies for lymphomas, we compared the TME composition of MYC-rearranged (MYC-R) high-grade B-cell lymphomas (HGBL) to ‘MYC negative’ diffuse large B-cell lymphomas, not otherwise specified (DLBCL) at DNA, RNA and protein levels. Methods Biopsy specimens from HGBL patients (HOVON-152 trial, NCT03620578) and DLBCL (HOVON-902 cohort, NCT04139252) were selected based on sample availability. MYC-R, BCL2-R and BCL6-R were determined using fluorescence in situ hybridization (FISH), using break-apart probes according to standard diagnostic lymphoma work up. We performed genomic profiling (targeted next-generation sequencing (tNGS), lon Torrent; n=36 HGBL/26 DLBCL) using the validated AmpliSeq BLYMFv2 panel, gene-expression profiling (GEP, NanoString; n=53/80) using the BLYMF777 probe set as previously described (PMID 34478526/35454765), and imaging mass cytometry (IMC, Hyperion; n=40/32) using a 41 antibody-marker panel for phenotyping to define the molecular landscape of tumors and determine the composition of their TME. Statistical differences between abundances were determined using the Fisher's exact test and Student's t-test. Results The majority of HGBL cases exhibited recurrent BLC2 rearrangements: 66% of cases in the genomic profiling and GEP cohorts and 62,5% in the IMC cohort were classified as ‘BCL2 double hit (DH)‘. BCL6 DH was observed in 11-15% of HGBL cases. Additionally, 21-22% of all HGBL cases had MYC, BCL2 and BCL6 rearrangements and were classified as triple hit. MYC-R were absent in all DLBCL cases. DNA: genomic profiling Consistent with previous literature, chromatin modifiers CREBBP and KMT2D were frequently mutated in HGBL as well as in DLBCL. Notably, HGBL had more mutations in TNFRSF14 (HVEM, p=0.007), BCL6 (p=0.01), and B-cell transcription factor IRF8 (p=0.04), while DLBCL showed higher frequencies of mutations in ZEB2 (p=0.01), KLHL6, GRHPR, IRF4 (all p=0.03) and tumor suppressor BTG2 (p=0.05), indicating distinct mutational profiles outside previously defined genetic subgroups (PMID 29713087/29641966). RNA: GEP signatures The majority of both HGBL and DLBCL cases were found to be GCB according to COO (Lymph2Cx). Despite mutational differences between the cohorts, GEP revealed a higher expression of genes related to the dark zone signature (DZsig, PMID 36302166/37552496) and MYC activity (PMID 27923830) in HGBL, while these genes were expressed at low levels in DLBCL. Protein: IMC phenotypes Using IMC we determined the composition of TME immune phenotypes in four cellular compartments: tumor, lymphoid, myeloid and stromal cells. In total, 666.809 cells (range 3-18K per patient) were obtained and 131 unique cellular phenotypes were defined. Clustering IMC phenotypes revealed three TME types: lymphoid immune-rich (mainly DLBCL cases), lymphoid immune-depleted (largely HGBL cases), and intermediate (including both DLBCL and HGBL). In detail, HGBL cases exhibited lower percentages of lymphoid cells (p<0.001) and were depleted for CD8+ T-cells, including naïve, effector memory and central memory CD8+ cytotoxic T-cells (all p<0.001), suggesting an immunosuppressed microenvironment. The amount of FOXP3+ T-cells (p=0.04), γδ-T-cells (p=0.03), and innate (like) lymphocytes (p<0.001) was also decreased in HGBL as compared to DLBCL. The TME of HGBL was significantly depleted in (proinflammatory) monocytes (p<0.04) and PD-L1+ macrophages (p<0.001), while there was a trend toward lower percentages of total myeloid cells in HGBL (p=0.07). Tumor and stromal cell percentages were similar between both groups (p=0.3). Conclusion This large comparison of MYC-R HGBL versus ‘MYC negative’ DLBCL cases using multi-omic analysis revealed a distinct TME in HGBL patients with increased frequencies of mutations in B-cell transcription factors, an abundance of discriminative MYC-related gene-expression signatures and, most strikingly, significantly lower infiltration of immune cells, especially cytotoxic T-cells, as compared to DLBCL. This suggests that MYC is linked to an immune-suppressed TME. These differences should be taken into account when developing T-cell engaging therapies for optimal efficacy.

Background ADAMTS-13 adopts an open conformation in patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) in acute phase while being closed in healthy donors. We reported that a substantial number of patients with iTTP in remission with restored ADAMTS-13 activity (>50%) still had an open ADAMTS-13 conformation, although a closed conformation is expected given the extent of remission. Objectives To investigate whether open ADAMTS-13, represented by a conformation index >0.5, is associated with a risk of earlier ADAMTS-13 and/or clinical relapse. Methods We collected follow-up data (ADAMTS-13 parameters, ADAMTS-13 and clinical relapse, and treatment) from 81 patients with iTTP in remission with ADAMTS-13 activity >50%. Results During follow-up, 19 ADAMTS-13 and 10 clinical relapses were reported (median follow-up period, 20 months). First, open or closed ADAMTS-13 conformation was dichotomized based on the 0.5 conformation index cutoff. Open ADAMTS-13 (conformation index, >0.5) was not identified as a risk factor for ADAMTS-13 and clinical relapse (log-rank test and Cox regression model). In contrast, by identifying the optimal conformation index cutoff for relapse prediction, using classification and regression tree analysis, a conformation index >0.645 and >0.835 was shown to be a risk factor for ADAMTS-13 relapse (hazard ratio, 3.3; 95% CI, 1.3-8.3; P = .01) and clinical relapse (hazard ratio, 4.4; 95% CI, 1.3-15.3; P = .02), respectively. Conclusion Patients with open ADAMTS-13 with a conformation index >0.645 and >0.835 have a >3- and >4-fold higher risk of earlier ADAMTS-13 and clinical relapse, respectively. Hence, ADAMTS-13 conformation index could be used to complement ADAMTS-13 activity monitoring to timely notice ADAMTS-13 relapse and prevent clinical relapse.

Background: There is ambiguity whether frail patients with atrial fibrillation (AF) managed with vitamin K antagonists (VKAs) should be switched to a non-vitamin K oral anticoagulant (NOAC). Methods: We conducted a pragmatic, multicenter, open-label, randomized controlled superiority trial. Older AF patients living with frailty (age ≥75 years plus a Groningen Frailty Indicator (GFI) score ≥3) were randomized to switch from INR-guided VKA treatment to a NOAC or to continued VKA treatment. Patients with a glomerular filtration rate <30 mL/min/1.73 m 2 or with valvular AF were excluded. Follow-up was 12 months. The cause-specific hazard ratio (HR) was calculated for occurrence of the primary outcome which was a major or clinically relevant non-major bleeding complication, whichever came first, accounting for death as a competing risk. Analyses followed the intention-to-treat principle. Secondary outcomes included thromboembolic events. Results: Between January 2018 and June 2022, a total of 2,621 patients were screened for eligibility and 1,330 patients were randomized (mean age 83 years, median GFI 4). After randomization 6 patients in the switch to NOAC arm and 1 patient in the continue with VKA arm were excluded due to the presence of exclusion criteria, leaving 662 patients switched from a VKA to a NOAC and 661 patients continued VKAs in the intention-to-treat population. After 163 primary outcome events (101 in the switch arm, 62 in the continue arm), the trial was stopped for futility according to a prespecified futility analysis. The HR for our primary outcome was 1.69 (95% CI 1.23-2.32). The HR for thromboembolic events was 1.26 (95% CI 0.60 to 2.61). Conclusions: Switching INR-guided VKA treatment to a NOAC in frail older patients with AF was associated with more bleeding complications compared to continuing VKA treatment, without an associated reduction in thromboembolic complications.

Von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. We developed a V HH-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in TTP patients during acute attacks of thrombotic microangiopathy, but not in remission. Finally, we show that therapeutic plasminogen activation in a mouse model for TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.

Introduction Patients (pts) with high grade B-cell lymphoma (HGBL) with MYC and BCL2 and/or BCL6 gene rearrangements (double hit and triple hit (DH/TH)) have poor outcomes to standard R-CHOP. Retrospective studies reported improved disease-free survival (DFS) through intensification with dose-adjusted etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab (DA-EPOCH-R). This regimen was studied in a small prospective trial including 24 DH/TH and 19 single MYC rearranged (SH) lymphomas (Dunleavy, Lancet Haematol 2019). No prospective studies evaluating DA-EPOCH-R exclusively for pts with DH/TH HGBL have been reported. The HOVON-152 trial aimed to improve outcomes in untreated DH/TH HGBL pts by investigating the efficacy of the immune checkpoint inhibitor nivolumab as consolidation treatment in pts achieving complete metabolic response (CMR) after DA-EPOCH-R induction. Here, we present the efficacy and safety profile of the induction phase with DA-EPOCH-R. Methods HOVON-152 is a prospective, multi-center, single arm phase II trial. Inclusion criteria were pts with newly diagnosed HGBL-DH/TH (according to the WHO 2016 classification), age ≥ 18 years, WHO performance status (PS) 0-3 and Ann Arbor stage II-IV. During the screening period for rearrangement status, pts could receive 1 cycle of R-CHOP or DA-EPOCH-R, followed by 5 cycles of DA-EPOCH-R. All pts received intrathecal CNS prophylaxis. All diagnostic lymphoma samples were centrally reviewed. PET-CT scans were performed at diagnosis, after 3 cycles and at end-of-induction (local review, central review will be reported at the conference). Pts in CMR after induction treatment (Deauville 1-3 or a negative lymphoma biopsy in case of Deauville 4) proceeded to nivolumab consolidation (480 mg every 4 weeks for one year). The HOVON-152 aimed to improve 12 months DFS of pts in CMR after induction from an expected 70% to 85% with nivolumab consolidation. Secondary objectives included evaluation of response rates, overall survival (OS) and safety. With a power of 0.90 a sample size of 97 pts was calculated. Here, we report efficacy (CMR rate) and safety of DA-EPOCH-R induction treatment. Logistic univariate analysis is used to analyze baseline characteristics associated with response. Adverse events (AEs) were defined according to the common terminology criteria for adverse events (CTC AE 5.0), counted by the highest grade per system organ class per patient. Results From August 2018 - March 2022, 97 pts have been enrolled (study inclusion completed). One patient was excluded due to CNS localization. The median age was 62 years (range 35-79); 90 (83%) pts had stage III-IV disease and 52 (54%) had (intermediate-)high international prognostic index (IPI) (Table 1). Central pathology review confirmed DH/TH in all pts. 65 pts (67%) had a BCL2 DH, 11 (12%) a BCL6 DH and 16 (17%) a TH. Dose adjustments were performed conform protocol. The maximum dose-level (DL) achieved was DL1 in 41 (43%), DL2 in 22 (23%), DL3 in 25 (25%), DL4 in 7 (7%) and DL5 in 1 (1%) of the pts. Vincristine dose was reduced in 28/81 (35%) pts. After DA-EPOCH-R induction, 63/96 (66%, 95% CI 55-75%) pts achieved CMR. WHO PS 2-3 (odds ratio (OR) 0.15, 95% CI 0.03-0.85, p=0.03), elevated LDH (OR 0.24, 95%CI 0.10-0.59, p=0.002) and bulky disease defined as ≥10 cm mass (OR 0.23, 95%CI 0.09-0.56, p=0.001), were significantly associated with a lower chance of achieving CMR. During treatment, 7 (7%) pts experienced a grade 5 AE (ileus, intestinal perforation, multi-organ failure/sepsis). Thereof, 4 patients died during DA-EPOCH-R and 3 patients died after DA-EPOCH-R. 31 (32%) experienced a grade 4 AE (e.g. sepsis, perforation, hemorrhage, thrombocytopenia and neutropenia), 21 (22%) a grade 3 AE (e.g. anemia, mucositis, infections and electrolyte disturbances) and 16 (17%) pts a grade 2 AE. Conclusion We report the largest prospective series of DH/TH HGBL patients treated with DA-EPOCH-R. DA-EPOCH-R induction was feasible, with toxicity and dose adjustments as previously described. The observed CMR rate of 66% was lower than previously reported in a prospective cohort of mixed DH/TH and SH patients (74%). For patients achieving CMR, the nivolumab consolidation phase is ongoing. Translational side studies investigating predictive factors to identify patients not achieving CMR are ongoing. For these patients, novel strategies to improve first-line treatment are warranted.

Background Thrombopoietin receptor agonists are frequently used in treating immune thrombocytopenia (ITP) owing to high response rates and good tolerability. ITP is associated with an increased risk of thrombosis. Whether treatment with eltrombopag further increases this risk is controversial. The mechanisms behind the thrombotic risk in ITP are unclear. Objectives To assess platelet function and hypercoagulability in patients with ITP and the effect of eltrombopag thereon. Methods This prospective multicenter study assessed adult primary patients with ITP who were starting eltrombopag treatment. Platelet (re)activity and hypercoagulability were measured in whole blood or plasma before start and after 2 to 3 weeks of eltrombopag treatment and compared with those of controls. Change over time was assessed by mixed-effects models, and the results were corrected for multiple testing. Results We included 16 patients and 33 controls. At baseline, patients with ITP exhibited lower expression of glycoprotein VI, more activated platelets, and lower reactivity toward agonists compared with controls. β-Thromboglobulin levels reduced and thrombin generation peak height increased compared with those of controls. In line with this finding, patients with ITP showed high factor VIII (median, 217%; IQR, 174%-272%) and von Willebrand factor levels (median, 167%; IQR, 109%-198%). Eltrombopag treatment increased thrombin generation potential: lag time decreased and peak height and endogeneous thrombin potential increased. The latter changes were not significant after correction for multiple testing. Conclusion Patients with ITP in this study were in a hypercoagulable state, with preactivated platelets, increased thrombin generation potential, and increased levels of factor VIII and von Willebrand factor. Eltrombopag treatment further increased plasma thrombin generation potential but no other hemostatic parameters.

Introduction The application of circulating cell-free DNA (cfDNA) has attracted recent interest for cancer detection and monitoring. The presence of somatic copy number alterations (SCNAs) can be used to differentiate between cancer cases and healthy controls (PMID: 29109393). This information can be retrieved from low-coverage whole genome sequencing (lcWGS) and used to determine the proportion of tumor derived cfDNA (ctDNA) in a tube of blood at a reasonable cost. Previous studies observed that cancer patients tend to have a higher fraction of shorter cfDNA fragments compared to healthy controls, irrespective of the presence of genomic events (PMID: 30404863). Here, we evaluate if a combination of cfDNA genomic and epigenomic features that can be retrieved from a single run of lcWGS, has potential for the early prediction of end of induction (EoI) response, in patients with high-grade lymphoma with MYC and BCL2 and/or BLC6 rearrangements (HGBL-DH/TH). Methods In the HOVON-152 phase II trial (NCT03620578) HGBL-DH/TH patients were treated with induction immunochemotherapy with one cycle of R-CHOP followed by five cycles of DA-EPOCH-R. Patients in complete metabolic response (CMR, defined as Deauville score 1-3) at the end of induction receive 1-year nivolumab consolidation. Blood was collected from 40 patients using PAXgene ccfDNA tubes after one cycle (timepoint 1, T1), three (T2) and six (T3) cycles of treatment. At T3, response to induction treatment was determined using 18F-FDG-PET scan. Patients with CMR at EoI were considered responders. In this study, we included 40 out of 97 patients from the HOVON-152 phase II trial, and enriched for patients with no CMR. Plasma was separated using dual centrifugation protocol, and cfDNA was isolated using the QIASymphony kit (QIAGEN). Sequencing libraries were prepared using the ThruPLEX Plasma-seq kit (Takara). lcWGS was performed on a Novaseq 6000 (Illumina). Somatic copy number aberrations (SCNAs) were retrieved with ichorCNA, insert size profiles were generated using Picard tools. SCNAs were classified as detected when the tumor fraction quantified with ichorCNA exceeded 3%. Results We assessed the detection of SCNAs and the SCNA-derived tumor fraction using the ichorCNA software. The ctDNA tumor fraction was found to be significantly increased in non-responders (NR, n=24) compared to responders (R, n=16), irrespective of sampling timepoint (Wilcoxon, p<0.001). At T1, the tumor fraction was significantly elevated in non-responders (Wilcoxon, p=0.014), and SCNAs were detected in 6/16 (38%) of the responders versus 19/24 (79%) of the non-responders, with negative predictive value (NPV): 71%, positive predictive value (PPV): 74%. Therefore, non-responders tend to have a higher fraction of tumor-derived signal which is already apparent at earlier timepoints. To evaluate the potential of using cfDNA fragmentation for the early detection of response, we retrieved the fragment size profiles. The proportion of short fragments (P20-150 bp), was found to be increased in non-responders compared to responders at T1 (Wilcoxon, p<0.001) and T2 (Wilcoxon, p=0.033), which suggests that the plasma of non-responders contains a higher fraction of tumor-signal (Figure 1A). Previous reports leveraged the size properties of ctDNA in order to increase the sensitivity of SCNA based methods (PMID: 30404863). In order to utilize both epigenomic and genomic properties to increase the detection rate of tumor-signal, we performed in silico size selection to computationally filter out fragments in the 20-150bp size range. Upon repeating the SCNA analysis, we observed that 20/24 (83%) non-responders and 1/15 (19%) responders had detectable ctDNA at T1 (Figure 1B). This yields a NPV and PPV of 80% and 86% respectively for EoI response detection at T1. By leveraging the fragmentomic properties of cfDNA, the sensitivity of SCNA-based methods can be improved, which leads to an increased detection of response at earlier timepoints. Discussion In patients with HGBL-DH/TH, after one cycle of R-CHOP, the presence of SCNAs enhanced by fragment size analysis in plasma, is significantly associated with an unfavourable EoI response, with a NPV and PPV of 80% and 86% respectively. This novel approach can be performed without prior knowledge of genetic alterations, and may have high potential to guide early risk-adapted treatment strategies in HGBL-DH/TH. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction The R2 regimen (lenalidomide-rituximab) is effective in treatment-naive and relapsed/refractory follicular lymphoma (R/R FL). Combining R2 with bendamustine (R2B) is feasible (Stevens WBC et al., Hemasphere 2020) and hypothesized to deepen remission and prolong event-free survival (EFS) compared to historic controls. We performed a randomized phase II trial of R2 and R2B, aiming at identifying the most promising arm to take forward to a randomized phase III trial. Methods In the multicenter, prospective, randomized, non-comparative phase II part of the HOVON110/ReBeL study (Dutch Clinical Trial Register NTR3028), patients (pts) with FL relapsed after ≤5 prior therapies were randomized 1:1 to R2 (Arm A) or R2B (Arm B). Stratification factors used were FLIPI score (0-2 vs 3-5), number of prior treatments (1 vs 2-5), prior bendamustine use and relapse during rituximab maintenance (RM). Arm A pts received 6 cycles of R2 q28 days (lenalidomide 20 mg day 1-21, rituximab 375 mg/m2 day 1); Arm B pts received 6 cycles of R2B q28 days (lenalidomide 20 mg day 3-21, rituximab 375 mg/m2 day 1, bendamustine 90 mg/m2 day 1,2). In both arms, pts with partial or complete remission (PR/CR) received 2 years of RM treatment, once every three months. Subcutaneous rituximab was allowed. Thrombosis prophylaxis was advised during induction; antimicrobial prophylaxis with valaciclovir and cotrimoxazole was mandatory in arm B. Arms A and B were separately evaluated for efficacy and toxicity. Co-primary endpoints were CT-based CR rate according to Cheson 2007 criteria at end of induction (EOI) and incidence of severe toxicities (ST), defined as grade ≥ 3 non-hematological toxicity, specified grade 4 hematologic toxicity (neutropenia lasting ≥ 7 days despite GCSF, febrile neutropenia, thrombocytopenia) or non-lymphoma related death. An event for event-free survival was defined as induction failure, progression, relapse or death from any cause. PET-CT was performed before and after induction. Central pathology and blinded central PET-CT review (Lugano classification) were performed. Results Between 2014 and 2019, 92 of 150 planned pts were randomized. The trial was stopped early because of slow accrual. Two pts (1 in each arm) were ineligible. Baseline characteristics were comparable between Arms A/B in terms of gender and median age (64/62 years). Most pts had stage III/IV disease (85%/80%), an intermediate/high risk FLIPI score (78%/89%) and had undergone 1 prior treatment (75%/76%; range 1-5). For pts in arms A/B, 80%/76% completed all 6 induction cycles and 41%/43% completed 8 cycles of RM. Main reasons for discontinuation during induction were progressive disease (PD) (n=4 per arm) and toxicity or other reasons in 5/7 patients. Severe toxicity (ST) occurred in 3 pts in arm A (6.8%; 2 fatal pneumonia cases) and in 6 pts in arm B (13.0%; no fatalities), while 43%/66% of pts experienced any grade 3-4 AE (2%/7% grade 4). These comprised mainly skin toxicity, infections and gastrointestinal toxicity. In the R2B arm, 1 pt had Pneumocystis carinii pneumonia during RM. Based on intention to treat (ITT) and local CT assessment (efficacy co-primary endpoint), 16% of R2 and 22% of R2B treated pts achieved a CR at EOI; the overall response rate (ORR) was 70% and 72%, respectively. The complete metabolic remission rate (CMR) by central PET-CT review at EOI was 48% and 54%. At a median follow-up of 48 months (mo), EFS was 39% and 61%; median EFS was 24.6 mo and not reached (NR). Importantly, median time to next treatment (TTnT) was 9.2 mo and NR. There were 7 second primary malignancies (SPM) in 5 pts in Arm A) and 5 SPM in 4 pts in Arm B). Overall survival was excellent in both arms with 72% and 91% of pts alive at 48 months. Causes of death during the study were progression of FL (n=8) and toxicity (n=3) in Arm A; FL (n=3), toxicity (n=1) and suicide (n=1) in Arm B. Conclusions This randomized non-comparative phase II trial of R2 and R2B in R/R FL patients showed high CT-based OR rates and PET-CT CMR rates, but low CT-based CR rates for both treatments. At a median FU of 48 months, 39% of R2 treated patients and 61% of R2B treated patients were still alive and in remission; this compares favorably with historic real world controls (Batlevi et al, Blood Cancer J 2020). Both regimens were associated with less severe toxicity than expected (pre-set acceptability threshold of 20% ST), supporting further investigation of R2 combinations in randomized phase III trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Introduction Treatment of patients with high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) with intensified immune-chemotherapy DA-EPOCH-R results in a 48-month event-free survival of 71.0% [Dunleavy, Lancet Haematol 2018]. In the HOVON-152, we investigate the added value of immune checkpoint PD-1 inhibition for patients who achieve complete metabolic remission (CMR) after DA-EPOCH-R induction. Nonetheless, whether DA-EPOCH-R has an effect on the immune system, and - reversely - whether the composition of immune system influences DA-EPOCH-R therapy are not known. To gain more insight on these important issues, we performed longitudinal high-throughput immune profiling of patients during the DA-EPOCH-R induction phase of the HOVON-152. Methods In the HOVON-152 single arm, phase II trial (NCT03620578) HGBL-DH/TH patients received 1 cycle of R-CHOP followed by 5 cycles of DA-EPOCH-R induction treatment. Peripheral blood was sampled after one cycle of R-CHOP (enrollment), before start of the 3rd DA-EPOCH-R cycle (midterm) and after the last DA-EPOCH-R cycle (end-of-induction, EOI). Patients achieving CMR at EOI (Deauville score 1-3 after induction) were identified as responders and proceeded with nivolumab consolidation (480 mg iv every 4 weeks) for 1 year. For the profiling we selected 55 patients (32 responders and 23 non-responders, enriched for non-responders) who completed the whole study. T and NK cells were enumerated through quantitative, dual platform flow cytometry of unseparated full blood. The frequencies of NK and T cell subsets were determined in cryopreserved PBMC through multiparameter flow cytometry. The high-throughput flow cytometry data were analyzed by computational methods UMAP and FlowSOM. Non-parametric methods were used for statistical testing. Results DA-EPOCH-R had no apparent effects on the total number of NK cells. The frequency of cytotoxic CD56dim NK cells was, however, gradually decreased during DA-EPOCH-R (p<0.001), consistent with rituximab-mediated consumption. There was also a gradual decrease in NK cells expressing maturation markers CD57 (p=0.001), TIGIT (p=0.009) and KLRG1 (p=0.027) and a transient decrease in DNAM1 (p<0.001). Consistently, the proportion of CD56bright CD16dim non-cytotoxic NK cells and the frequency of NK cells expressing inhibitory receptor NKG2A became more abundant (all p<0.001). Despite losing the cytotoxic CD56dimphenotype, the NK cells showed activation induced alterations such as the progressive increase of NKp30 (p<0.001) and transient increase of HLA-DR (p<0.001) during therapy. More interestingly, DA-EPOCH-R appeared to have significant impact on T cells: while total T cell frequencies and numbers showed only a temporary decrease at midterm, a progressive decrease in the CD4/CD8 ratio (p=0.016) and a progressive increase in the expression of T cell activation markers CD127 (p<0.01) and CD38 (p=0.004) was clearly visible. HLA-DR, another activation marker, also showed an increase at EOI (p=0.01) after a transient decrease at midterm (p=0.01) (Figure 1A). The analysis of exhaustion markers TIM3, LAG3 and TIGIT revealed a transient increase of LAG3 on CD8 cells (p≤0.018). Most remarkably, DA-EPOCH-R treatment was significantly associated with a progressive decline in the frequency of PD-1 positive CD8 T cells (p=0.037) (Figure 1B). Also interestingly, the responding patients tended to have lower PD-1 levels on their T cells at the end of treatment as compared to non-responders (p=0.052). Further analyses regarding the possible impact of immune system on DA-EPOCH-R outcome revealed that a (relative) abundance of non-cytotoxic CD56bright NK cells (p=0.006) and higher CD3 T cells (p=0.04) at enrollment was associated with achievement of CMR. Conclusion In conclusion, treatment of HGBL-DH/TH patients with DA-EPOCH-R results not only in the expected rituximab-mediated alterations in the NK cell compartment, but also influences the T cell compartment with a shift towards a lower CD4/CD8 ratio, more T cell activation and a reduction of PD-1 expression on CD8 T cells. Higher T cell frequencies at baseline and decreased frequencies of PD-1+ CD8 T cells at EOI were furthermore associated with achievement of CMR. Overall, these data contribute to a wider understanding of NK and T cell dynamics during DA-EPOCH-R and points to an considerable involvement of T cells in therapy outcome. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Acquired thrombotic thrombocytopenic purpura (aTTP) is a thrombotic microangiopathy with a severe mortality and morbidity. Caplacizumab has recently been approved in the Netherlands as a new therapeutic option in patients with life-threatening organ failure due to aTTP. We describe the case of a 50 year old patient with aTTP who was referred to our hospital for treatment with caplacizumab. After undergoing treatment with plasmapheresis, prednisolone, rituximab and caplacizumab, her platelet count recovered and she was ready to be discharged. Unfortunately, before discharge she developed a fatal intra-cerebral hemorrhage. Fatal hemorrhage as an adverse event of caplacizumab has not been described before. Up to now there is no evidence-based treatment for caplacizumab induced heavy bleeding.

Background: The bispecific antibody blinatumomab (blina) is approved for patients(pts) with relapsed/refractory precursor B-cell ALL (ALL). It has also shown to be highly effective in MRD+ pts in first line treatment. TABLE - Pt characteristics Total, N(%) 71(100%) Age(y), median(range) 53(18-70) Age (y), N(%) ≤40 22(31%) 40-60 29(41%) >60 20(28%) BM blasts (%), N(%) ≤50% 4(6%) >50% 62(87%) Unknown 5(7%) Karyotype, N(%) Ph+ 26/71(37%) KMT2A 2/67(3%) Complex 23/67(34%) Hypodiploidy 5/67(7%) Aims: To evaluate whether blina added in upfront therapy to prephase and after consolidation (cons)-1 would increase MRD negativity measured by qPCR or flowcytometry at a cut-off of <10-4 (primary endpoint). Secondary endpoints included CR, EFS, OS, adverse events and treatment-related mortality. Methods: Pts, 18-70 years(y) old, with newly diagnosed CD19+ ALL (incl. Ph+), were included. Treatment was based on a pediatric inspired protocol (HOVON 70, Rijneveld et al., 2011) with reduced doses of anthracyclines, MTX, etoposide and PEG-ASP for pts ≥40y old. Prephase consisted of 10 days steroids, from day 5 combined with 14 days blina in the standard step-up dosing schedule. After cons-1 and after intensification 2, two 4-week blina courses were added irrespective of MRD. The protocol was amended twice due to toxicity. First, in 2018 the first PEG-ASP administration was omitted. Second, in 2021 doxorubicin, dexamethasone and PEG-ASP were reduced during intensification 1. Rituximab (if CD20+), prophylactic ITs and imatinib (if Ph+) were standard. AlloHSCT was offered to intermediate and high-risk pts. Trial was registered with ClinicalTrials.gov, identifier NCT03541083. Results: Seventy-one pts were enrolled. Pt characteristics are presented (Table). Fifteen pts discontinued treatment before blina cons-1 due to refractory disease (n=8), toxicity (n=7) or death (n=2). In the total study population, 55/71 pts (77%) achieved CR after (blina) cons-1. Among pts still on treatment after cons-1, 55/56 (98%) pts achieved CR, 50/55 (91%) reached MRD negativity. After prephase, CR was already reached in 63% and MRD negativity in 53%. Blina related AEs in prephase were as expected (83% of pts had ≥1 AE and 10% had ≥1 SAE (hepatotoxicity 3 pts, pain lymph node 1, CRS 1, pneumonia 1, renal insufficiency 1)). CRS was observed in 35% of pts, 32% of whom experienced grade 3 and no grade ≥4. During prephase 5 pts discontinued blina; during blina cons-1, 4 pts stopped blina. With a median follow-up of 17,6 months, the estimated 2-y EFS was 64% standard error (SE) ± 7% (≤60y 71% SE ± 9% and >60y 47% SE ± 12%). Overall, 14 (20%) pts died. OS after 2y was 73% SE ± 7% (≤60y 82% SE ± 8% and >60y 52% SE ± 14%) (Figure 1). For pts with Ph+ ALL, 2-y EFS was 88% SE ± 6%and OS also 88% SE ± 7%. For Ph- ALL, 2-y EFS and OS were 53% SE ± 9% and 68% SE ± 9%, resp. Among pts who reached CR on protocol (n=60), 5 (8%) had relapse, 6 (10%) died and 6 (10%) discontinued treatment due to toxicity. Until now, 22 pts proceeded to alloHSCT and 11 with maintenance. Image:Summary/Conclusion: Blina can safely be added to prephase of an intensified pediatric schedule for newly diagnosed ALL up to 70y of age, albeit with dose reductions for PEG-ASP, doxorubicin and dexamethasone. The combination increases CR and MRD negativity rate. The early addition of blina resulted in very early achievement of MRD negativity with 53% after prephase and 91% after blina cons-1. Further reductions of chemotherapy should be explored (especially for Ph+) if these results are maintained with longer follow-up.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with thrombosis. We conducted a cohort study of consecutive patients, suspected of SARS-CoV-2 infection presented to the emergency department. We investigated haemostatic differences between SARS-CoV-2 PCR positive and negative patients, with dedicated coagulation analysis. The 519 included patients had a median age of 66 years, and 52.5% of the patients were male. Twenty-six percent of the patients were PCR-positive for SARS-CoV-2.PCR positive patients had increased levels of fibrinogen and (active) von Willebrand Factor (VWF) and decreased levels of protein C and α2-macroglobulin compared to the PCR negative patients. In addition, we found acquired activated protein C resistance in PCR positive patients. Furthermore, we found that elevated levels of factor VIII and VWF and decreased levels of ADAMTS-13 were associated with an increased incidence of thrombosis in PCR positive patients. In conclusion, we found that PCR positive patients had a pronounced prothrombotic phenotype, mainly due to an increase of endothelial activation upon admission to the hospital. These findings show that coagulation tests may be considered useful to discriminate severe cases of COVID-19 at risk for thrombosis.