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.

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.

Infectious complications after chemotherapy and stem cell transplantation in multiple myeloma: implications of Fc gamma receptor and myeloperoxidase promoter polymorphisms.

Multiple myeloma is associated with a high risk of infections. We hypothesized that Fc gamma receptor (FCGR) and myeloperoxidase (MPO) promoter gene polymorphisms influence the risk of infections after induction chemotherapy (IC) and autologous stem cell transplantation (ASCT). Retrospectively, we analysed 136 patient courses of IC and 113 procedures of ASCT. Genetic analyses were made with PCR techniques on genomic DNA. The incidence rate ratio of sepsis during ASCT in patients homozygous for the G-129MPO promoter type was 0.30 (95% CI: 0.09-0.96). The G-463AMPO promoter polymorphism was not associated with the risk of infections. The polymorphisms of FCGR2A, FCGR3A and FCGR3B were not convincingly associated with infections. The NA1 variant of FCGR3B was strongly skewed with other risk factors, and the results in IC and ASCT were conflicting. Further studies of the G-129AMPO promoter as a potential risk modifier for infections are relevant.

Rapid single-step methods for detection of two immune defence gene polymorphisms: the myeloperoxidase (MPO) G-129A and the Fc gamma receptor 2A (FCGR2A) H/R131.

Polymorphisms of immune defence genes may act as disease modifiers and are studied by many researchers. A conclusive analysis of the impact of genetic variations typically requires a large number of sample specimens, and in retrospective studies this may include samples of reduced quality, e.g. formalin-fixed paraffin-embedded tissue specimens. Here we describe two new single-step methods for rapid and sensitive analysis of: 1. The G-129A myeloperoxidase (MPO) promoter polymorphism, which affects the amount of myeloperoxidase in neutrophils. 2. The Fc gamma receptor 2A (FCGR2A)-H/R131 polymorphism, which is critical to the binding of IgG2 immune complexes to phagocytes.