Contributions
Type: Publication Only
Background
During B-cell development functional immunoglobulin genes are assembled from individual V, D, and J gene segments to generate V-D-J combinations of length and sequence that are unique for each cell. Rearrangements within IGH occur first, followed by IGK. Along with the Vk and Jk segments, there are other elements in the IGK loci that can be involved in recombination. The Kde can rearrange to Vk gene, but also to an isolated RSS in the Jk-Ck intron (INTR). If IGK rearrangements that produce functional IgH/IgK molecules are not successful, rearrangement proceeds within the other immunoglobulin light chain (IGL) locus.
Leukemia and lymphomas originating from the malignant transformation of individual lymphoid cells generally share one or more of these cell specific or “clonal” gene rearrangements. Assays that identify clonal lymphocyte populations in clinical specimens are used on a routine basis to assist in the diagnosis of lymphoproliferative disease.
Currently, most IGK clonality assays use multiplex PCR followed by capillary electrophoresis. However, these methods often do not provide sufficient analytical sensitivity and are incapable of identifying the specific rearranged DNA sequence data required to track clones in follow up testing. The emergence of cost-effective, next-generation sequencing (NGS) platforms and development with associated bioinformatics tools have resulted in powerful new approaches for clonality detection and monitoring.
Aims
To establish the performance of a sensitive, robust and reliable NGS assay formatted for the Illumina® MiSeq instrument that includes the bioinformatics tools to identify clonal IGK gene rearrangements and the corresponding specific V-J DNA sequence information.
Methods
LymphoTrack® IGK Assay (Invivoscribe, Inc., San Diego, CA USA) uses a single PCR multiplex master mix to amplify each sample (24 indices,5 reactions each, are included in each kit).The amplicons were purified using the AMPureXP system and purified equimolar amounts of amplicons were pooled to generate a library. A portion of the library was sequenced on a MiSeq instrument using the MiSeq v2 Reagent kit (500 cycles). The MiSeq data was analyzed using LymphoTrack® bioinformatics software, which generated frequency distributions and identified the rearranged DNA sequences.
Results
Data generated with the LymphoTrack® IGK Assay and bioinformatics software identified clonality and corresponding DNA sequences of IGK Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements. The results obtained generally were concordant with results of testing with capillary electrophoresis. The assay demonstrated excellent linearity, sensitivity, and reproducibility using contrived samples. This assay was used to test genomic DNA from peripheral blood, bone marrow and FFPE specimens. In addition, this NGS testing approach identified the specific break point sequence of the INTR element.
Summary
A comprehensive NGS assay has been developed for the Illumina MiSeq platform that identifies clonal IGK Vk-Jk, Vk-Kde and INTR-Kde rearrangements and the associated specific DNA sequences. The assay can be used both to detect and monitor clonal populations.
Keyword(s): B-CLL, Clonality, Diagnosis, Non-Hodgkin's lymphoma
Session topic: Publication Only
Type: Publication Only
Background
During B-cell development functional immunoglobulin genes are assembled from individual V, D, and J gene segments to generate V-D-J combinations of length and sequence that are unique for each cell. Rearrangements within IGH occur first, followed by IGK. Along with the Vk and Jk segments, there are other elements in the IGK loci that can be involved in recombination. The Kde can rearrange to Vk gene, but also to an isolated RSS in the Jk-Ck intron (INTR). If IGK rearrangements that produce functional IgH/IgK molecules are not successful, rearrangement proceeds within the other immunoglobulin light chain (IGL) locus.
Leukemia and lymphomas originating from the malignant transformation of individual lymphoid cells generally share one or more of these cell specific or “clonal” gene rearrangements. Assays that identify clonal lymphocyte populations in clinical specimens are used on a routine basis to assist in the diagnosis of lymphoproliferative disease.
Currently, most IGK clonality assays use multiplex PCR followed by capillary electrophoresis. However, these methods often do not provide sufficient analytical sensitivity and are incapable of identifying the specific rearranged DNA sequence data required to track clones in follow up testing. The emergence of cost-effective, next-generation sequencing (NGS) platforms and development with associated bioinformatics tools have resulted in powerful new approaches for clonality detection and monitoring.
Aims
To establish the performance of a sensitive, robust and reliable NGS assay formatted for the Illumina® MiSeq instrument that includes the bioinformatics tools to identify clonal IGK gene rearrangements and the corresponding specific V-J DNA sequence information.
Methods
LymphoTrack® IGK Assay (Invivoscribe, Inc., San Diego, CA USA) uses a single PCR multiplex master mix to amplify each sample (24 indices,5 reactions each, are included in each kit).The amplicons were purified using the AMPureXP system and purified equimolar amounts of amplicons were pooled to generate a library. A portion of the library was sequenced on a MiSeq instrument using the MiSeq v2 Reagent kit (500 cycles). The MiSeq data was analyzed using LymphoTrack® bioinformatics software, which generated frequency distributions and identified the rearranged DNA sequences.
Results
Data generated with the LymphoTrack® IGK Assay and bioinformatics software identified clonality and corresponding DNA sequences of IGK Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements. The results obtained generally were concordant with results of testing with capillary electrophoresis. The assay demonstrated excellent linearity, sensitivity, and reproducibility using contrived samples. This assay was used to test genomic DNA from peripheral blood, bone marrow and FFPE specimens. In addition, this NGS testing approach identified the specific break point sequence of the INTR element.
Summary
A comprehensive NGS assay has been developed for the Illumina MiSeq platform that identifies clonal IGK Vk-Jk, Vk-Kde and INTR-Kde rearrangements and the associated specific DNA sequences. The assay can be used both to detect and monitor clonal populations.
Keyword(s): B-CLL, Clonality, Diagnosis, Non-Hodgkin's lymphoma
Session topic: Publication Only