Mutations in the TP53 tumor suppressor gene occur at different frequencies in a variety of human malignancies.1 TP53 encodes a DNA-binding transcription factor that induces cell growth arrest, senescence and cell death by apoptosis upon cellular stress, including oncogenic stress and DNA damage. In hematological malignancies TP53 mutations are common in relapsed/refractory disease and confers a dismal prognosis.2 PRIMA-1 and the analog APR-246 (PRIMA-1MET) can restore wild-type conformation of mutant p53 and induce apoptosis in tumor cells of various origin.3, 4, 5 APR-246 is the first compound targeting mutant p53 to enter into a clinical trial. In the first in-man study APR-246 was given daily as a 2 h intravenous infusion for 4 consecutive days. The maximum tolerable dose using this schedule was defined as 60 mg/kg daily and the most common adverse effects were neurological.3 This is an extension of the first in-man study aiming at optimizing the dose regimen to obtain better antitumor response with less toxicity. The primary objective was to evaluate safety and tolerability of APR-246 with the new dosing schedule. Secondary objectives were to determine the pharmacokinetic (PK) profile and assess antitumor effects of APR-246. Main inclusion criteria were: relapsed/refractory non-M3 acute myeloid leukemia (AML) or chronic lymphatic leukemia (CLL) with deletion of 17p not eligible for other therapies, age 18 years, ECOG performance status 0–2 and life expectancy of at least 2 months. Main exclusion criteria were hepatic transferase 5 upper limit of normal (ULN), creatinine 1.5 ULN, severe cardiac or respiratory disease. The trial was conducted in accordance to the Declaration of Helsinki and ICH-GCP guidelines and was registered at ClinicalTrials.gov, identifier NCT00900614. The dosing schedule was designed to maximize drug exposure without increasing peak concentrations. Four daily infusions were given using a boosting infusion of 50 mg/kg during 45 min followed by a 85 mg/kg as a 5 h and 15 min infusion, in total 135 mg/kg, with an option to repeat the treatment in 21 day cycles. Interim analyses were scheduled at completion of every three patients or whenever necessary at any occasion. Dose limiting toxicity (DLT) was defined as study drug–related Common Terminology Criteria for Adverse Events (CTCAE) grade 1 for ataxia/incoordination, tremor and confusion; CTCAE grade 2 for somnolence, depressed level of consciousness and seizures; and other CTCAE grade 2, 3 or 4. Dose de-escalation was made twice by decision from the study board due to toxicity. After treating three patients on the starting dose of 135 mg/kg, there was a dose reduction to 105 mg/kg (45 mg/kg bolus+60 mg/kg infusion). This schedule was given to five patients when a second de-escalation was made to 67.5 mg/kg (25 mg/kg+42.5 mg/kg). Efficacy was assessed by blood and bone marrow sampling at baseline, on day 4 and on day 21 for AML, and by lymph node evaluation manually; and with computed tomography (CT) scans at baseline and on day 21 for CLL. Predefined response criteria for AML was a reduction of blast cell count of 25% in peripheral blood and/or bone marrow, and for CLL a reduction of lymphocytes in peripheral blood of 25% and/or a 25% reduction of tumor size assessed by CT scan or palpation of peripheral lymph nodules and/or disappearance of B symptoms. A Mini Mental Test was performed before and after infusion at day 1 and day 4. Safety visits twice weekly were made until day 21. PK samples were collected at baseline, 45 and 90 min after start and 2 h after completed infusion. PK calculations were performed by non-compartmental analysis. Plasma concentrations of APR-246 were determined using a validated LC-MS/MS method.3 Eleven patients were screened and ten were included at four centers, eight with AML and two with CLL. One patient failed screening due to an ongoing systemic infection and was not included. All patients had relapsed or refractory disease with a median of two previous lines of therapy (range 1–3). Three out of eight patients analyzed (exon 2–11) had TP53 mutations. Patient demographics are summarized in Table 1. Using the 6 h infusion administration, the PK of APR-246 was characterized by a low clearance (151±35 ml/h per kg) and a large distribution volume (824±219 ml/kg; mean±s.d., n=10) indicating a distribution of APR-246 throughout the whole body. APR-246 concentrations declined in plasma with a t1/2 of about 3.8±0.8 h. At steady state (day 4), no accumulation of APR-246 was observed with the adopted 6 h infusion schedule or after repeated cycles. Plasma clearance and distribution volume were similar at day 1 and day 4, suggesting time-independent kinetics, and in line with the PK parameters previously obtained in the range of doses 2–90 mg/kg after a 2 h infusion. This suggests dose-independent kinetics also up to a daily dose of 135 mg/kg per day given as a 6 h infusion. In total, 41 adverse events (AE) were considered to have probable or possible relationship to study treatment. There was a relation between the dose and the number of AE:s probably associated with study medication: 16, 11 and 1 AE:s were reported at dose levels 135, 105 and 67.5 mg/kg, respectively (Table 2). The most commonly reported AE were vomiting, constipation and dizziness, followed by nausea, electrocardiogram QT-prolongation, cough and fatigue. Two AE:s were classified as dose limiting at 135 mg/kg; dizziness, reported for the second included patient, and tremor, reported for the third included patient. Based on this, the dose for the following patients was decreased to 105 mg/kg. The only two severe adverse events (SAE) judged as probably related to the study medication occurred at this dose level, both in the same patient (03-102) and on the same day; electrocardiogram QT-prolongation and dyskinesia. Patient 03-102 was discontinued from the study due...

Cytogenetically normal acute myeloid leukemia (CN-AML) compose between 40% and 50% of all adult acute myeloid leukemia (AML) cases. In this clinically diverse group, molecular aberrations, such as FLT3-ITD, NPM1, and CEBPA mutations, recently have added to the prognostic accuracy. Aberrant DNA methylation is a hallmark of cancer, including AML. We investigated in total 118 CN-AML samples in a test and a validation cohort for genome-wide promoter DNA methylation with Illumina Methylation Bead arrays and compared them with normal myeloid precursors and global gene expression. IDH and NPM1 mutations were associated with different methylation patterns (P = .0004 and .04, respectively). Genome-wide methylation levels were elevated in IDH-mutated samples (P = .006). We observed a negative impact of DNA methylation on transcription. Genes targeted by Polycomb group (PcG) proteins and genes associated with bivalent histone marks in stem cells showed increased aberrant methylation in AML (P < .0001). Furthermore, high methylation levels of PcG target genes were independently associated with better progression-free survival (odds ratio = 0.47, P = .01) and overall survival (odds ratio = 0.36, P = .001). In summary, genome-wide methylation patterns show preferential methylation of PcG targets with prognostic impact in CN-AML.

A commonly deleted region in chronic lymphocytic leukemia (CLL) is the 11q22–23 region, which encompasses the ATM gene. Evidence suggests that tumor suppressor genes other than ATM are likely to be involved in CLL with del(11q). A microRNA (miR) cluster including the miR-34b and miR-34c genes is located, among other genes, within the commonly deleted region (CDR) at 11q. Interestingly, these miRs are part of the TP53 network and have been shown to be epigenetically regulated. In this study, we investigated the expression and methylation status of these miRs in a well-characterized cohort of CLL, including cases with/without 11q-deletion. We show that the miR-34b/c promoter was aberrantly hypermethylated in a large proportion of CLL cases (48%, 25/52 cases). miR-34b/c expression correlated inversely to DNA methylation (P = 0.003), and presence of high H3K37me3 further suppressed expression regardless of methylation status. Furthermore, increased miR-34b/c methylation inversely correlated with the presence ...

The British Journal of Haematology publishes original research papers in clinical, laboratory and experimental haematology. The Journal also features annotations, reviews, short reports, images in haematology and Letters to the Editor.

ACKR1/DARC-associated neutropenia (NP; ADAN; Online Mendelian Inheritance in Man 611862), caused by a variation in the ACKR1/DARC gene (rs2814778), is common in persons of African or Middle Eastern descent. In a cohort of 66 genetically confirmed subjects with ADAN, we show that absolute neutrophil counts (ANCs) may occasionally be lower than previously recognized (0.1 × 109-0.49 × 109/L for 9% of the subjects), which is similar to ANCs in severe congenital NP (SCNP). ANCs often normalized during inflammation, even mild. Individuals with ADAN (of 327 observed person-years) showed no cases of myelodysplastic syndrome (MDS), which is frequently encountered in SCNP. Unexpectedly, 22% presented with autoantibodies to neutrophils, compared with <1% in controls. Compared with healthy donors, subjects with ADAN demonstrated significantly lower human cationic antimicrobial protein-18/pro-leucin leucin-37 plasma levels; higher levels of nonclassical, proinflammatory, 6-sulfo LacNac-expressing monocytes; and differentially expressed plasma levels of 28 of the 239 analyzed cytokines related to immunity/inflammation, cell signaling, neutrophil activation, and angiogenesis. Collectively, more severe neutropenia in ADAN than previously assumed may complicate differential diagnoses compared with other SCNPs, and various (auto)immune/inflammatory reactions with a distinct profile may be a cause or consequence of this hereditary neutropenia.