Erythroid predominance in bone marrow biopsies of AML patients after decitabine treatment correlates with mutation profile and complete remission.

INTRODUCTION: Acute myeloid leukemia (AML) patients may receive hypomethylating agents (HMAs) such as decitabine (DAC) as part of their treatment. Not all patients respond to this therapy, and if they do the clinical response may occur only after 3 to 6 courses of treatment. Hence, early biomarkers predicting response would be very useful. METHODS: We retrospectively analyzed a cohort of 22 AML patients who were treated with DAC. Histology of the bone marrow biopsy, pathogenic mutations and methylation status were related to the treatment response. RESULTS: In 8/22 (36%) patients, an erythroid dominant response (EDR) pattern, defined as a ratio of myeloid cells/erythroid cells < 1, was observed. In the remaining 14 cases a myeloid predominance was preserved during treatment. No difference in the hypomethylating effect of DAC treatment was observed in patients with and without EDR, as global 5-methylcytosine levels dropped similarly in both groups. Mutational analysis by NGS using a panel of commonly mutated genes in AML, showed that patients with an early EDR harbored on average less mutations, with U2AF1 mutations occurring more frequently, whereas RUNX1 mutations were underrepresented compared to non-EDR cases. Interestingly, the development of an EDR correlated with complete remission (7/8 cases with an EDR versus only 2/14 cases without an EDR). CONCLUSION: We conclude that early histological bone marrow examination for the development of an EDR may be helpful to predict response in AML patients during treatment with DAC.

Oral Iptacopan Monotherapy in Paroxysmal Nocturnal Hemoglobinuria.

BACKGROUND: Persistent hemolytic anemia and a lack of oral treatments are challenges for patients with paroxysmal nocturnal hemoglobinuria who have received anti-C5 therapy or have not received complement inhibitors. Iptacopan, a first-in-class oral factor B inhibitor, has been shown to improve hemoglobin levels in these patients. METHODS: In two phase 3 trials, we assessed iptacopan monotherapy over a 24-week period in patients with hemoglobin levels of less than 10 g per deciliter. In the first, anti-C5-treated patients were randomly assigned to switch to iptacopan or to continue anti-C5 therapy. In the second, single-group trial, patients who had not received complement inhibitors and who had lactate dehydrogenase (LDH) levels more than 1.5 times the upper limit of the normal range received iptacopan. The two primary end points in the first trial were an increase in the hemoglobin level of at least 2 g per deciliter from baseline and a hemoglobin level of at least 12 g per deciliter, each without red-cell transfusion; the primary end point for the second trial was an increase in hemoglobin level of at least 2 g per deciliter from baseline without red-cell transfusion. RESULTS: In the first trial, 51 of the 60 patients who received iptacopan had an increase in the hemoglobin level of at least 2 g per deciliter from baseline, and 42 had a hemoglobin level of at least 12 g per deciliter, each without transfusion; none of the 35 anti-C5-treated patients attained the end-point levels. In the second trial, 31 of 33 patients had an increase in the hemoglobin level of at least 2 g per deciliter from baseline without red-cell transfusion. In the first trial, 59 of the 62 patients who received iptacopan and 14 of the 35 anti-C5-treated patients did not require or receive transfusion; in the second trial, no patients required or received transfusion. Treatment with iptacopan increased hemoglobin levels, reduced fatigue, reduced reticulocyte and bilirubin levels, and resulted in mean LDH levels that were less than 1.5 times the upper limit of the normal range. Headache was the most frequent adverse event with iptacopan. CONCLUSIONS: Iptacopan treatment improved hematologic and clinical outcomes in anti-C5-treated patients with persistent anemia - in whom iptacopan showed superiority to anti-C5 therapy - and in patients who had not received complement inhibitors. (Funded by Novartis; APPLY-PNH ClinicalTrials.gov number, NCT04558918; APPOINT-PNH ClinicalTrials.gov number, NCT04820530.).

Development of a target concentration intervention to individualize paroxysmal nocturnal hemoglobinuria treatment with pegcetacoplan.

Pegcetacoplan (Aspaveli®/Empaveli™) is a factor C3 inhibitor that is approved for the treatment of paroxysmal nocturnal hemoglobinuria. An individualized dosing strategy might be useful to improve patient-friendliness and cost-effectiveness of this very expensive drug. Therefore, the aim of this study was to develop an individualized treatment regimen for pegcetacoplan based on the pharmacokinetic-pharmacodynamic data of the manufacturer. We conducted a clinical trial simulation with the approved dosing regimen of 1080 mg twice-weekly and a target concentration intervention-based dosing regimen in patients with and without prior eculizumab use. For eculizumab-naïve patients, the target concentration intervention-based dosing regimen resulted in a comparable fraction of patients with LDH normalization (LDH < 226 U/L) and hemoglobulin normalization (> 12 g/dL) compared to the approved regimen (LDH 50.2% and 50.0% respectively and hemoglobulin 45.6% and 44.4%). A modest dose reduction of ~ 5% was possible with target concentration intervention-based dosing. An intensified dosing interval was necessary in 2.3% of the patients however an interval prolongation was possible in 28.2% of the patients. Similar results were obtained for patients prior treated with eculizumab. In this study we show the potential of an individualized dosing regimen of pegcetacoplan with can improve patient friendliness in approximately 30% of the patients and improve therapy in approximately 2% of the patients at slightly reduced costs.

Identification and surveillance of rare relapse-initiating stem cells during complete remission post-transplantation.

Relapse following complete remission (CR) remains the main cause of mortality after allogeneic stem cell transplantation for hematological malignancies and therefore improved biomarkers for early prediction of relapse remains a critical goal towards development and assessment of preemptive relapse treatment. Since the significance of cancer stem cells as a source of relapses remains unclear, we investigated whether mutational screening for persistence of rare cancer stem cells would enhance measurable residual disease (MRD) and early relapse-prediction post-transplantation. In a retrospective study of relapse patients and continuous-CR patients with myelodysplastic syndromes and related myeloid malignancies, combined flow cytometric cell sorting and mutational screening for persistence of rare relapse-initiating stem cells was performed in bone marrow at multiple CR time points post-transplantation. In 25 CR samples from 15 patients that later relapsed, only 9 samples were MRD-positive in mononuclear cells (MNCs) whereas flowcytometric sorted hematopoietic stem and progenitor cells (HSPCs) were MRD-positive in all samples, and always with a higher variant allele frequency than in MNCs (mean 97-fold). MRD-positivity in HSPCs preceded MNCs in multiple sequential samples, in some cases preceding relapse by more than 2 years. In distinction, in 13 patients in long-term continuous-CR, HSPCs remained MRD-negative. Enhanced MRD-sensitivity was also observed in total CD34+ cells, but HSPCs were always more clonally involved (mean 8-fold).In conclusion, identification of relapse-initiating cancer stem cells and mutational MRD-screening for their persistence consistently enhances MRD-sensitivity and earlier prediction of relapse after allogeneic stem cell transplantation.

Proteinuria and Exposure to Eculizumab in Atypical Hemolytic Uremic Syndrome.

BACKGROUND: Eculizumab is a monoclonal antibody for the treatment of atypical hemolytic uremic syndrome (aHUS). Kidney damage, a common condition in patients with aHUS, may result in proteinuria. Because proteinuria may affect the pharmacokinetics of therapeutic proteins such as eculizumab, the aim of our study was to investigate the effect of proteinuria on eculizumab pharmacokinetics. METHODS: This study was an ancillary study of a previously performed pharmacokinetic-pharmacodynamic study of eculizumab in aHUS. Proteinuria, measured as urinary protein-creatinine ratios (UPCR), was investigated as covariate for eculizumab clearance. Thereafter, we evaluated the effect of proteinuria on the exposure to eculizumab in a simulation study for the initial phase and for a 2-weekly and 3-weekly interval in the maintenance phase. RESULTS: The addition of UPCR as a linear covariate on clearance to our base model resulted in a statistically improved fit ( P < 0.001) and reduction of unexplained variability in clearance. From our data, we predicted that in the initial phase, 16% of the adult patients with severe proteinuria (UPCR >3.1 g/g) will have inadequate complement inhibition (classical pathway activity >10%) on day 7 of treatment, compared with 3% of the adult patients without proteinuria. None of the pediatric patients will have inadequate complement inhibition at day 7 of treatment. For the 2- and 3-weekly dosing intervals, we predicted that, respectively, 18% and 49% of the adult patients and, respectively, 19% and 57% of the pediatric patients with persistent severe proteinuria will have inadequate complement inhibition, compared with, respectively, 2% and 13% of the adult patients and, respectively, 4% and 22% of the pediatric patients without proteinuria. CONCLUSIONS: Severe proteinuria is associated with a higher risk of underexposure to eculizumab. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: CUREiHUS, Dutch Trial Register, NTR5988/NL5833.

Nationwide study of eculizumab in paroxysmal nocturnal hemoglobinuria: Evaluation of treatment indications and outcomes.

Eculizumab is an effective treatment for paroxysmal nocturnal hemoglobinuria (PNH). However, considering the risk of life-threatening meningococcal disease, life-long duration and costs, there are strict criteria for initiation of therapy. To evaluate the application and real-world effectiveness of eculizumab in the Netherlands, a multicenter retrospective cohort study was conducted: indications and treatment outcomes were collected for 105 Dutch PNH patients. In all patients, eculizumab was initiated conforming to indications as formulated in the Dutch PNH guideline. According to recently published response criteria, 23.4% of the patients had reached a complete hematological response, 53.2% a good or partial response, and 23.4% a minor response after 12 months of therapy. In the majority of patients the response remained stable during long-term follow-up. The degree and relevance of extravascular hemolysis significantly differed between response groups (p = 0.002). Improvements of EORTC-QLQc30 and FACIT-fatigue scores were observed, however patients reported lower scores than the general population. A detailed evaluation of 18 pregnancies during eculizumab showed no maternal or fetal deaths, and no thromboembolic events during pregnancy. This study demonstrates that the majority of patients benefit from eculizumab when adhering to the indications as formulated in the Dutch PNH guideline. However, novel therapies are needed to further improve real-world outcomes, such as hematological responses and quality of life.

Dose optimalization of subcutaneous ravulizumab is predicted to yield significant savings and to improve patient friendliness.

Ravulizumab is an expensive complement C5-inhibitor for the treatment of paroxysmal nocturnal haemoglobinuria. Recently, a subcutaneous formulation has entered the market, for which the approved dosing regimen results in supratherapeutic ravulizumab concentrations in the majority of patients in the registration studies. Therefore, we explored alternative dosing regimens in silico based on the registration data of the manufacturer. Extending the interval from 1 to 2 weeks or individualized dosing based on therapeutic drug monitoring resulted in therapeutic ravulizumab concentrations and comparable predicted efficacy in terms of lactate dehydrogenase normalization, with dose reductions up to 64%. We here show that with an individualized dose, a substantial dose reduction for subcutaneous ravulizumab might be possible with improved patient-friendliness.

Anti Thymocyte Globulin-Based Treatment for Acquired Bone Marrow Failure in Adults.

Idiopathic acquired aplastic anemia can be successfully treated with Anti Thymocyte Globulin (ATG)-based immune suppressive therapy and is therefore considered a T cell-mediated auto immune disease. Based on this finding, several other forms of idiopathic acquired bone marrow failure are treated with ATG as well. For this review, we extensively searched the present literature for evidence that ATG can lead to enduring remissions in different forms of acquired multi- or single-lineage bone marrow failure. We conclude that ATG-based therapy can lead to an enduring hematopoietic response and increased overall survival (OS) in patients with acquired aplastic aplasia. In patients with hypocellular myelodysplastic syndrome, ATG can lead to a hematological improvement without changing the OS. ATG seems less effective in acquired single-lineage failure diseases like Pure Red Cell Aplasia, Amegakaryocytic Thrombocytopenia and Pure White Cell Aplasia, suggesting a different pathogenesis in these bone marrow failure states compared to aplastic anemia. T cell depletion is hypothesized to play an important role in the beneficial effect of ATG but, as ATG is a mixture of polyclonal antibodies binding to different antigens, other anti-inflammatory or immunomodulatory effects could play a role as well.

The potential of individualized dosing of ravulizumab to improve patient-friendliness of paroxysmal nocturnal haemoglobinuria treatment at reduced costs.

Ravulizumab is a very expensive complement C5-inhibitor for the treatment of paroxysmal nocturnal haemoglobinuria, with a fixed-dosing interval of 8 weeks. For lifelong treatment, a cost-effective and patient-friendly dosing strategy is preferred. We therefore explored alternative ravulizumab dosing regimens in silico based on the thorough dose-finding studies of the manufacturer. Extending the interval to 10 weeks or individually extending the interval to a mean of 12.8 weeks based on pharmacokinetic monitoring resulted in noninferior efficacy in terms of lactate dehydrogenase normalization, with drug cost savings up to 37%. We here show the potential of individualized ravulizumab dosing to improve patient-friendliness at reduced costs.

Neutrophil specific granule and NETosis defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder characterized by a lack of α-granules in platelets and progressive myelofibrosis. Rare loss-of-function variants in neurobeachin-like 2 (NBEAL2), a member of the family of beige and Chédiak-Higashi (BEACH) genes, are causal of GPS. It is suggested that BEACH domain containing proteins are involved in fusion, fission, and trafficking of vesicles and granules. Studies in knockout mice suggest that NBEAL2 may control the formation and retention of granules in neutrophils. We found that neutrophils obtained from the peripheral blood from 13 patients with GPS have a normal distribution of azurophilic granules but show a deficiency of specific granules (SGs), as confirmed by immunoelectron microscopy and mass spectrometry proteomics analyses. CD34+ hematopoietic stem cells (HSCs) from patients with GPS differentiated into mature neutrophils also lacked NBEAL2 expression but showed similar SG protein expression as control cells. This is indicative of normal granulopoiesis in GPS and identifies NBEAL2 as a potentially important regulator of granule release. Patient neutrophil functions, including production of reactive oxygen species, chemotaxis, and killing of bacteria and fungi, were intact. NETosis was absent in circulating GPS neutrophils. Lack of NETosis is suggested to be independent of NBEAL2 expression but associated with SG defects instead, as indicated by comparison with HSC-derived neutrophils. Since patients with GPS do not excessively suffer from infections, the consequence of the reduced SG content and lack of NETosis for innate immunity remains to be explored.

Patient preferences and quality of life implications of ravulizumab (every 8 weeks) and eculizumab (every 2 weeks) for the treatment of paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Eculizumab has transformed management of paroxysmal nocturnal hemoglobinuria (PNH) since its approval. However, its biweekly dosing regimen remains a high treatment burden. Ravulizumab administered every 8 weeks demonstrated noninferiority to eculizumab in two phase 3 trials. In regions where two PNH treatment options are available, it is important to consider patient preference. OBJECTIVE: The aim of this study was to assess patient preference for ravulizumab or eculizumab. METHODS: Study 302s (ALXN1210-PNH-302s) enrolled PNH patients who participated in the extension period of phase 3 study ALXN1210-PNH-302. In the parent study, eculizumab-experienced adult PNH patients received ravulizumab or eculizumab during a 26-week primary evaluation period. All patients in the extension period received ravulizumab. In study 302s, patient treatment preference was evaluated using an 11-item PNH-specific Patient Preference Questionnaire (PNH-PPQ©). Of 98 patients, 95 completed PNH-PPQ© per protocol for analysis. RESULTS: Overall, 93% of patients preferred ravulizumab whereas 7% of patients either had no preference (6%) or preferred eculizumab (1%) (P < 0.001). For specific aspects of treatment, ravulizumab was preferred (in comparison to no preference or eculizumab) on infusion frequency (98% vs. 0% vs. 2%), ability to plan activities (98% vs. 0% vs. 2%), and overall quality of life (88% vs. 11% vs. 1%), among other aspects. Most participants selected frequency of infusions as the most important factor determining preference (43%), followed by overall quality of life (23%). CONCLUSION: This study shows that a substantial proportion of patients preferred ravulizumab over eculizumab and provides an important patient perspective on PNH treatment when there is more than one treatment option.

Clinical and biological impact of TET2 mutations and expression in younger adult AML patients treated within the EORTC/GIMEMA AML-12 clinical trial.

We assessed the prognostic impact of TET2 mutations and mRNA expression in a prospective cohort of 357 adult AML patients < 60 years of age enrolled in the European Organization For Research and Treatment of Cancer (EORTC)/Gruppo Italiano Malattie Ematologiche dell' Adulto (GIMEMA) AML-12 06991 clinical trial. In addition the co-occurrence with other genetic defects and the functional consequences of TET2 mutations were investigated. TET2 mutations occurred in 7.6 % of the patients and were an independent marker of poor prognosis (p = 0.024). TET2 and IDH1/2 mutations strongly associated with aberrations in the DNA methyltransferase DNMT3A. Functional studies confirmed previous work that neither nonsense truncations, nor missense TET2 mutations, induced 5-hydroxymethylcytosine formation. In addition, we now show that mutant TET2 forms did not act in a dominant negative manner when co-expressed with the wild-type protein. Finally, as loss-of-function TET2 mutations predicted poor outcome, we questioned whether low TET2 mRNA expression in cases of AML without TET2 mutations would affect overall survival. Notably, also AML patients with low TET2 mRNA expression levels showed inferior overall survival.

Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes.

In myelodysplastic syndromes (MDS), deletions of chromosome 7 or 7q are common and correlate with a poor prognosis. The relevant genes on chromosome 7 are unknown. We report here that EZH2, located at 7q36.1, is frequently targeted in MDS. Analysis of EZH2 deletions, missense and frameshift mutations strongly suggests that EZH2 is a tumor suppressor. As EZH2 functions as a histone methyltransferase, abnormal histone modification may contribute to epigenetic deregulation in MDS.

Acquired mutations in TET2 are common in myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of neoplastic hematopoietic disorders. Several recurrent chromosomal aberrations have been associated with MDS, but the genes affected have remained largely unknown. To identify relevant genetic lesions involved in the pathogenesis of MDS, we conducted SNP array-based genomic profiling and genomic sequencing in 102 individuals with MDS and identified acquired deletions and missense and nonsense mutations in the TET2 gene in 26% of these individuals. Using allele-specific assays, we detected TET2 mutations in most of the bone marrow cells (median 96%). In addition, the mutations were encountered in various lineages of differentiation including CD34(+) progenitor cells, suggesting that TET2 mutations occur early during disease evolution. In healthy tissues, TET2 expression was shown to be elevated in hematopoietic cells with highest expression in granulocytes, in line with a function in myelopoiesis. We conclude that TET2 is the most frequently mutated gene in MDS known so far.