Subject(s)
Bone Marrow Cells/cytology , Bone Marrow/metabolism , Endothelial Progenitor Cells/cytology , Stem Cells/cytology , Adult , Bone Marrow/drug effects , Bone Marrow Cells/drug effects , Cells, Cultured , Endothelial Progenitor Cells/drug effects , Humans , Male , Middle Aged , Neoplasms/drug therapy , Stem Cells/drug effectsABSTRACT
INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed. METHODS: DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance. RESULTS: An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected. CONCLUSION: In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.
Subject(s)
Biomarkers, Tumor , High-Throughput Nucleotide Sequencing , Leukemia, Myeloid, Acute/diagnosis , Leukemia, Myeloid, Acute/genetics , Alleles , DNA Mutational Analysis/methods , DNA Mutational Analysis/standards , Gene Frequency , High-Throughput Nucleotide Sequencing/methods , High-Throughput Nucleotide Sequencing/standards , Humans , Mutation , Reagent Kits, Diagnostic/standards , Reproducibility of ResultsABSTRACT
Analysis of short tandem repeats (STR) is the predominant method for post-transplant monitoring of donor engraftment. It can enable early detection of disease relapse, level of engraftment and provide useful information on the graft-versus-host disease (GVHD)/graft-versus-tumour (GVT) effect, facilitating therapeutic intervention. Harmonization and standardization of techniques and result interpretation is essential to reduce the impact of laboratory variability on both clinical management and the results of multi-centre clinical trials. However, the United Kingdom National External Quality Assessment Service for Leucocyte Immunophenotyping (UK NEQAS LI) has highlighted significant issues inherent in STR testing that impact upon inter- and intra- laboratory variation. We present here consensus best practice guidelines and recommendations for STR chimerism testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 11 UK and Eire clinical laboratories. This document uses data obtained from the UK NEQAS LI Post-Stem Cell Transplant (SCT) Chimerism Monitoring Programme.
Subject(s)
Chimerism , Hematopoietic Stem Cell Transplantation , Transplantation Chimera , Genetic Testing/methods , Genetic Testing/standards , Humans , Microsatellite Repeats , Transplantation Chimera/genetics , Transplantation, Homologous , Watchful WaitingABSTRACT
Molecular testing for the BCR-ABL1 fusion gene by real time quantitative polymerase chain reaction (RT-qPCR) is the most sensitive routine approach for monitoring the response to therapy of patients with chronic myeloid leukaemia. In the context of tyrosine kinase inhibitor (TKI) therapy, the technique is most appropriate for patients who have achieved complete cytogenetic remission and can be used to define specific therapeutic milestones. To achieve this effectively, standardization of the laboratory procedures and the interpretation of results are essential. We present here consensus best practice guidelines for RT-qPCR testing, data interpretation and reporting that have been drawn up and agreed by a consortium of 21 testing laboratories in the United Kingdom and Ireland in accordance with the procedures of the UK Clinical Molecular Genetics Society.
