Droplet digital PCR for sensitive relapse detection in acute myeloid leukaemia patients transplanted by reduced intensity conditioning.

INTRODUCTION: Follow-up after allogeneic transplantation in acute myeloid leukaemia (AML) is guided by measurable residual disease (MRD) testing. Quantitative polymerase chain reaction (qPCR) is the preferred MRD platform but unfortunately, 40%-60% of AML patients have no high-quality qPCR target. This study aimed to improve MRD testing by utilising droplet digital PCR (ddPCR). ddPCR offers patient-specific monitoring but concerns of tracking clonal haematopoiesis rather than malignant cells prompt further validation. METHODS: Retrospectively, we performed MRD testing on blood and bone marrow samples from AML patients transplanted by reduced-intensity conditioning. RESULTS: The applicability of ddPCR was 39/42 (92.9%). Forty-five ddPCR assays were validated with a 0.0089% median sensitivity. qPCR targeting NPM1 mutation detected relapse 46 days before ddPCR (p = .03). ddPCR detected relapse 34.5 days before qPCR targeting WT1 overexpression (p = .03). In non-relapsing patients, zero false positive ddPCR MRD relapses were observed even when monitoring targets associated with clonal haematopoiesis such as DNMT3A, TET2, and ASXL1 mutations. CONCLUSION: These results confirm that qPCR targeting NPM1 mutations or fusion transcripts are superior in MRD testing. In the absence of such targets, ddPCR is a promising alternative demonstrating (a) high applicability, (b) high sensitivity, and (c) zero false positive MRD relapses in non-relapsing patients.

Mitochondrial cytochrome c oxidase subunit II variations predict adverse prognosis in cytogenetically normal acute myeloid leukaemia.

Alterations in the two catalytic genes cytochrome c oxidase subunits I and II (COI and COII) have recently been suggested to have an adverse impact on prognosis in patients with acute myeloid leukaemia (AML). In order to explore this in further detail, we sequenced these two mitochondrial genes in diagnostic bone marrow or blood samples in 235 patients with AML. In 37 (16%) patients, a non-synonymous variation in either COI or COII could be demonstrated. No patients harboured both COI and COII non-synonymous variations. Twenty-four (10%) patients had non-synonymous variations in COI, whereas 13 (6%) patients had non-synonymous variations in COII. The COI and COII are essential subunits of cytochrome c oxidase that is the terminal enzyme in the oxidative phosphorylation complexes. In terms of disease course, we observed that in patients with a normal cytogenetic analysis at disease presentation (CN-AML) treated with curative intent, the presence of a non-synonymous variation in the COII was an adverse prognostic marker for both overall survival and disease-free survival (DFS) in both univariate (DFS; hazard ratio (HR) 4.4, P = 0.006) and multivariate analyses (DFS; HR 7.2, P = 0.001). This is the first demonstration of a mitochondrial aberration playing an adverse prognostic role in adult AML, and we argue that its role as a potentially novel adverse prognostic marker in the subset of CN-AML should be explored further.

Genetic and epigenetic similarities and differences between childhood and adult AML.

BACKGROUND: The biology of acute myeloid leukemia (AML) is complex and includes both genetic and epigenetic aberrations. We addressed the combined consequences of promoter hypermethylation of p15, CDH1, ER, MDR1, and RARB2 and mutation of NPM1, CEBPA, FLT3, and WT1 in a Danish cohort of 70 pediatric and 383 adult AML patients. PROCEDURE: Mutation analysis was done by fragment analysis followed by sequencing or by sequencing alone. Methylation status was determined using methylation-sensitive melting curve analysis (MS-MCA) after initial bisulfite modification. RESULTS: Among pediatric AMLs, we found promoter hypermethylation in p15 (47%), CDH1 (64%), ER (62%), MDR1 (8%), and RARB2 (22%) and mutations in NPM1 (11%), CEBPA (3%), FLT3ITD (4%), FLT3D835 (7%), and WT1 (7%). Promoter hypermethylation was significantly more frequent in core binding factor leukemias (CBF) compared to AMLs with abnormalities involving 11q23 (P = 0.024). Compared to adult AML we found a significant difference in p15 (47% vs. 73%, P < 0.001) and RARB2 (22% vs. 42%, P = 0.003) methylation, as well as in NPM1 (11% vs. 31%, P = 0.001) and FLT3ITD (4% vs. 26%, P < 0.001) mutation. CONCLUSION: Age-related differences exist in the frequency of mutations and it appears that promoter hypermethylation occurs in a non-random pattern in childhood AML accompanying specific genetic aberrations, and might represent an important step in the leukemogenic transformation.

A patient with a 20-year lag phase between JAK2-V617F+ myeloproliferation and NPM1-mutated AML arguing against a common origin of disease.

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.

Sepsis in acute myeloid leukaemia patients receiving high-dose chemotherapy: no impact of chitotriosidase and mannose-binding lectin polymorphisms.

Infections after chemotherapy often cause significant morbidity in patients with acute myeloid leukaemia (AML). Chitotriosidase (CHIT) and mannose-binding lectin (MBL) are part of the innate immune system. Polymorphism in the CHIT-coding gene (CHIT1) may be associated with Gram-negative sepsis in children with AML, and polymorphism in the MBL-coding gene (MBL2) seems to modify the risk of infections in several patient groups. The purpose of this study was to investigate the possible associations between polymorphisms in CHIT1, MBL2 and sepsis in adult patients treated with high-dose chemotherapy for AML. We included 190 patients treated with 526 cycles of chemotherapy. The follow-up period was 6 months from the diagnosis of AML. Prophylactic antibiotics were not used. We identified 604 febrile episodes with 246 episodes of sepsis. Thirty-two patients (17%) either died from infection or infection was a major concomitant factor for death. No significant associations between CHIT1 polymorphism and sepsis (P = 0.85) or death caused by sepsis (P = 0.14) were found. Furthermore, no significant associations between MBL2 polymorphism and sepsis (P = 0.76) or death caused by sepsis (P = 0.24) were observed. The severe and long-lasting neutropenia and mucositis after chemotherapy may explain why the MBL system does not protect against sepsis in patients with AML. Replacement therapy with recombinant MBL is not likely to decrease the risk of sepsis in patients with AML.

The combined expression of HOXA4 and MEIS1 is an independent prognostic factor in patients with AML.

HOXA4 gene expression is a predictor for outcome in normal karyotypic acute myeloid leukaemia (AML) patients. Given that Meis1 is a co-factor for Hox genes, we hypothesized that the combined expression of HOXA4 and MEIS1 might add prognostic information in these patients. When diagnostic samples from 246 AML patients were divided into three main groups based on gene expression levels of HOXA4 combined with MEIS1 we found that within the group of patients exhibiting low levels of HOXA4, those with a high expression of MEIS1 had a significantly worse outcome than those exhibiting low MEIS1 expression (P = 0.025). Moreover, this prediction was independent of cytogenetics, mutational status of the NPM1 and FLT3 genes as well as upon WBC and age. To evaluate the possible contribution of regulatory events underlying these observations, 157 patient samples were subjected to promoter hypermethylation analysis. We observed that 77% were HOXA4- and 15%MEIS1 hypermethylated and that this epigenetic alteration was highly correlated to the gene expression level (MEIS1: P = 0.001; HOXA4: P = 0.007). Finally, we found a higher expression level and a higher frequency of hypermethylation of HOXA4 among patients with NPM1 mutations. In conclusion, our data show that the combination of low HOXA4 and low MEIS1 gene expression is a favourable predictor for outcome in all AML patients and that the expression levels are governed by the methylation state of these genes.

Capillary gel electrophoresis: a simple method for identification of mutations and polymorphisms in the CEBPA gene in patients with acute myeloid leukaemia.

INTRODUCTION: The CEBPA gene encodes a transcription factor, CCAAT/enhancer binding protein (C/EBP)alpha. Expression of the wildtype protein is essential for the lineage specific differentiation of myelocytic haematopoietic precursors into mature neutrophils. Eight percentage of all AML patients harbour at least one mutation in this gene, increasing up to 15% in the group, where standard karyotypic analysis do not reveal chromosomal aberrations. OBJECTIVE: We designed a method, which discriminates as little as single base insertions or deletions accounting for 90% of all CEBPA mutations. The TAD2C length polymorphism was also identified using this set up. PATIENTS AND METHODS: Diagnostic bone marrow or peripheral blood from 446 adults and 39 children diagnosed with AML from 1980 to 2006 was analysed for mutations by PCR and capillary gel electrophoresis. RESULTS: We analysed pretreatment samples from 485 AML patients and 57 healthy volunteers and identified sequence variations in 35/446 adults and 1/39 children. We were immediately able to distinguish N- and C-terminal insertions and deletions as well as normally occurring polymorphisms. Abnormal PCR products were reprocessed and analysed by direct sequencing. We found stringent accordance between the two methods and reached the same frequency of mutations and polymorphisms as known from the literature. CONCLUSION: We conclude that capillary gel electrophoresis can be used as an accurate and high throughput diagnostic procedure for mutational status in the CEBPA gene identifying not only the same mutational frequency as in the published reports but also the TAD2C polymorphism in addition.

Gene expression profiling of Polycomb, Hox and Meis genes in patients with acute myeloid leukaemia.

The Polycomb group (PcG) of genes is important for differentiation and cell-cycle regulation and is aberrantly expressed in several cancers. To analyse the role of deregulated PcG genes in acute myeloid leukaemia (AML), we determined by RQ-PCR the expression of the PcG genes BMI-1, MEL18, SCML2, YY1 and EZH2, and the downstream PcG targets HOXA4, HOXA9 and MEIS1 in diagnostic bone marrow samples from 126 AML patients. There was a general overexpression of the genes in AML patients compared to 20 healthy donors, except of HOXA4 and MEL18, which both displayed a wide range of expression levels within the AML subgroups. Among the AML patients with normal karyotype, a low HOXA4 level was associated with a shorter overall survival (P = 0.005). In addition, expression levels of MEL18 and EZH2 were significantly (P < 0.025) higher in patients with complex karyotype and lower in CBF-mutated patients. The t(8;21) vs. inv(16) positive patients showed significantly different expression of SCML2, BMI-1, YY1, HOXA9 and MEIS1 (P < or = 0.01). Comparisons between the PcG and PcG-regulated genes and a number of clinical and molecular data revealed correlations to genes involved in DNA methylation (DNMT1, DNMT3B), apoptosis (BAX, CASPASE 3) and multidrug-resistance (MDR1, MRP ) (P < 0.01). In conclusion, our data suggest that the role of PcG and PcG-regulated genes in leukaemogenesis varies between, as well as within karyotypic subgroups.

Promoter hypermethylation of the retinoic acid receptor beta2 gene is frequent in acute myeloid leukaemia and associated with the presence of CBFbeta-MYH11 fusion transcripts.

Silencing of the putative tumour suppressor gene retinoic acid receptor beta2 (RARbeta2) caused by aberrant promoter hypermethylation has been identified in several solid tumours. In order to evaluate the extent of RARbeta2 hypermethylation and transcription in acute myeloid leukaemia (AML) at diagnosis, 320 patients were investigated by bisulphite-denaturing gradient gel electrophoresis and mRNA transcription levels were analysed in 61 of these by quantitative real-time polymerase chain reaction. The results were compared with demographic- and molecular data from the patients. While RARbeta2 was unmethylated in 10/10 bone marrow and 7/7 blood samples from healthy individuals, the gene was hypermethylated in 43% of the AML patients. The RARbeta2 degree of promoter methylation differed between and within individuals, and the mRNA transcription levels of the gene varied inter-individually by a factor of 4000. A significant inverse correlation between promoter hypermethylation and gene expression could be established (t-test, P = 0.019). Comparison of methylation data with a series of other molecular alterations in the same patient materials revealed a correlation between hypermethylation of the RARbeta2 promoter and the presence of CBF-MYH11 fusion transcripts (P < 0.01). Our data suggest that RARbeta2 promoter methylation is frequent in AML and may co-operate with the expression of CBF-MYH11 fusion transcripts in leukaemogenesis.