Contributions
Abstract: S801
Type: Oral Presentation
Presentation during EHA22: On Sunday, June 25, 2017 from 08:30 - 08:45
Location: Room N103
Background
Amplicon-based next generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements can be used to identify suitable markers for subsequent quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Within the EuroClonality-NGS Consortium we established and validated a standardized quality controlled amplicon-based NGS application to detect clonally rearranged IGH, IGK, TRB, TRG and TRD genes in lymphoid disorders.
Aims
Methods
Within the EuroClonality-NGS Consortium, V, D, and J gene-specific primers tailed with universal and T7-linker sequences, were designed to amplify complete and incomplete IGH, IGK, TRB, TRG or TRD gene rearrangements employing eight different multiplex PCR assays. PCR protocols were standardized in a common NGS workflow for all targets. NGS assays were tested in a European multi-laboratory pilot run in five institutes (Kiel, Bristol, Prague, Monza, Paris). Diagnostic DNA (100ng) from 10 ALL patients per centre were amplified in each institute using the EuroClonality NGS primer sets, and subsequently sequenced on the Illumina MiSeq (2×250bp v2 kit). Defined copy numbers of clonal reference DNAs were spiked into all samples for calculation of clonotype copy numbers. A standard polytarget quality control (QC) DNA was used to monitor inter- and intra-lab NGS reproducibility. Results of NGS based marker identification were compared to results of routine SS results.
Results
Conclusion
The IG / TR NGS panel, as established by the EuroClonality-NGS Consortium, allows for quality controlled high-throughput detection of clonal IG /TR rearrangements in ALL. Compared to low throughput methods more clonal MRD markers are identified, sensitivity is increased, processing time is reduced and labour intensive conventional methods to resolve mixed sequences in case of bi-allelic rearrangements or background are avoided.
Session topic: 1. Acute lymphoblastic leukemia - Biology
Keyword(s): Molecular markers, Minimal residual disease (MRD), Ig and TCR gene rearrangement, Acute lymphoblastic leukemia
Abstract: S801
Type: Oral Presentation
Presentation during EHA22: On Sunday, June 25, 2017 from 08:30 - 08:45
Location: Room N103
Background
Amplicon-based next generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements can be used to identify suitable markers for subsequent quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). Within the EuroClonality-NGS Consortium we established and validated a standardized quality controlled amplicon-based NGS application to detect clonally rearranged IGH, IGK, TRB, TRG and TRD genes in lymphoid disorders.
Aims
Methods
Within the EuroClonality-NGS Consortium, V, D, and J gene-specific primers tailed with universal and T7-linker sequences, were designed to amplify complete and incomplete IGH, IGK, TRB, TRG or TRD gene rearrangements employing eight different multiplex PCR assays. PCR protocols were standardized in a common NGS workflow for all targets. NGS assays were tested in a European multi-laboratory pilot run in five institutes (Kiel, Bristol, Prague, Monza, Paris). Diagnostic DNA (100ng) from 10 ALL patients per centre were amplified in each institute using the EuroClonality NGS primer sets, and subsequently sequenced on the Illumina MiSeq (2×250bp v2 kit). Defined copy numbers of clonal reference DNAs were spiked into all samples for calculation of clonotype copy numbers. A standard polytarget quality control (QC) DNA was used to monitor inter- and intra-lab NGS reproducibility. Results of NGS based marker identification were compared to results of routine SS results.
Results
Conclusion
The IG / TR NGS panel, as established by the EuroClonality-NGS Consortium, allows for quality controlled high-throughput detection of clonal IG /TR rearrangements in ALL. Compared to low throughput methods more clonal MRD markers are identified, sensitivity is increased, processing time is reduced and labour intensive conventional methods to resolve mixed sequences in case of bi-allelic rearrangements or background are avoided.
Session topic: 1. Acute lymphoblastic leukemia - Biology
Keyword(s): Molecular markers, Minimal residual disease (MRD), Ig and TCR gene rearrangement, Acute lymphoblastic leukemia