A NGS IGK ASSAY FOR THE DETECTION OF CLONALITY AND THE MONITORING OF MINIMAL RESIDUAL DISEASE
(Abstract release date: 05/19/16)
EHA Library. Xie Z. 06/09/16; 132516; E967
Dr. Zhiyi Xie
Contributions
Contributions
Abstract
Abstract: E967
Type: Eposter Presentation
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. In addition to V-D-J rearrangement, the IGK Kde locus can rearrange to Vk gene and to an isolated RSS in the Jk-Ck intron (INTR).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. The clinical relevance of minimal residual disease (MRD) detection in lymphoproliferative disease has been demonstrated by many studies. However, allele-specific primers are required for the qPCR-based MRD monitoring.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 MRD monitoring without the requirement of allele-specific designs.
Aims
To establish an IGK NGS assay that, combined with an IGH NGS assay, will increase the chance of identifying clonal populations and provide easy tracking of identified clonal population for MRD in lymphoid malignancies.
Methods
Genomic DNA from cell line, peripheral blood, bone marrow aspirates, and FFPE were tested for IGK rearrangements using the LymphoTrack® IGK MiSeq® Assay (Invivoscribe Inc., San Diego, USA). The IGK libraries were run individually or in combination with IGH and or TRG libraries. The sequencing data was analyzed using LymphoTrack® bioinformatics software, which performs the separation of IGK, IGH, and TRG data as necessary, and generates frequency distributions and identifies the rearranged DNA sequences for each target. Total of 198 peripheral blood samples were also tested by the capillary electrophoresis (CE) based IdentiClone® IGK assay.
Results
Data generated with the IGK NGS assay and bioinformatics software identified clonality and corresponding DNA sequences of IGK Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements. Excellent linearity of the IGK NGS assay was obtained from contrived cell line DNA in the range of 2.5 – 20% when running individually (R2=0.9897) or in combination with IGH and TRG assays (R2=0.9905). The concordance between the 198 samples tested by IGK NGS and CE assays was 77%. There were 43 samples indentified to be clonal out of 198 samples tested by both IGK and IGH NGS assays. Among those samples, 31 and 25 samples were clonal for IGK and IGH, respectively, and 18 samples were clonal for both IGK and IGH. It was demonstrated that the NGS assay allows for easy tracking of clonal populations at the sensitivity of 10-4 or at even lower limits of dectection, provided sufficient amount of DNA is tested.
Conclusion
A comprehensive assay has been developed for the MiSeq® platform that identifies clonal IGK rearrangements and the associated specific DNA sequences. The IGK assay, in combination with the IGH assay, increased the detection of clonality by 72% comparing to IGH alone. The assay can be used both to detect clonality and monitor the identified clonal populations in MRD.
Session topic: E-poster
Keyword(s): B-CLL, Clonality, Diagnosis, Non-Hodgkin's lymphoma
Type: Eposter Presentation
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. In addition to V-D-J rearrangement, the IGK Kde locus can rearrange to Vk gene and to an isolated RSS in the Jk-Ck intron (INTR).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. The clinical relevance of minimal residual disease (MRD) detection in lymphoproliferative disease has been demonstrated by many studies. However, allele-specific primers are required for the qPCR-based MRD monitoring.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 MRD monitoring without the requirement of allele-specific designs.
Aims
To establish an IGK NGS assay that, combined with an IGH NGS assay, will increase the chance of identifying clonal populations and provide easy tracking of identified clonal population for MRD in lymphoid malignancies.
Methods
Genomic DNA from cell line, peripheral blood, bone marrow aspirates, and FFPE were tested for IGK rearrangements using the LymphoTrack® IGK MiSeq® Assay (Invivoscribe Inc., San Diego, USA). The IGK libraries were run individually or in combination with IGH and or TRG libraries. The sequencing data was analyzed using LymphoTrack® bioinformatics software, which performs the separation of IGK, IGH, and TRG data as necessary, and generates frequency distributions and identifies the rearranged DNA sequences for each target. Total of 198 peripheral blood samples were also tested by the capillary electrophoresis (CE) based IdentiClone® IGK assay.
Results
Data generated with the IGK NGS assay and bioinformatics software identified clonality and corresponding DNA sequences of IGK Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements. Excellent linearity of the IGK NGS assay was obtained from contrived cell line DNA in the range of 2.5 – 20% when running individually (R2=0.9897) or in combination with IGH and TRG assays (R2=0.9905). The concordance between the 198 samples tested by IGK NGS and CE assays was 77%. There were 43 samples indentified to be clonal out of 198 samples tested by both IGK and IGH NGS assays. Among those samples, 31 and 25 samples were clonal for IGK and IGH, respectively, and 18 samples were clonal for both IGK and IGH. It was demonstrated that the NGS assay allows for easy tracking of clonal populations at the sensitivity of 10-4 or at even lower limits of dectection, provided sufficient amount of DNA is tested.
Conclusion
A comprehensive assay has been developed for the MiSeq® platform that identifies clonal IGK rearrangements and the associated specific DNA sequences. The IGK assay, in combination with the IGH assay, increased the detection of clonality by 72% comparing to IGH alone. The assay can be used both to detect clonality and monitor the identified clonal populations in MRD.
Session topic: E-poster
Keyword(s): B-CLL, Clonality, Diagnosis, Non-Hodgkin's lymphoma
Abstract: E967
Type: Eposter Presentation
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. In addition to V-D-J rearrangement, the IGK Kde locus can rearrange to Vk gene and to an isolated RSS in the Jk-Ck intron (INTR).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. The clinical relevance of minimal residual disease (MRD) detection in lymphoproliferative disease has been demonstrated by many studies. However, allele-specific primers are required for the qPCR-based MRD monitoring.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 MRD monitoring without the requirement of allele-specific designs.
Aims
To establish an IGK NGS assay that, combined with an IGH NGS assay, will increase the chance of identifying clonal populations and provide easy tracking of identified clonal population for MRD in lymphoid malignancies.
Methods
Genomic DNA from cell line, peripheral blood, bone marrow aspirates, and FFPE were tested for IGK rearrangements using the LymphoTrack® IGK MiSeq® Assay (Invivoscribe Inc., San Diego, USA). The IGK libraries were run individually or in combination with IGH and or TRG libraries. The sequencing data was analyzed using LymphoTrack® bioinformatics software, which performs the separation of IGK, IGH, and TRG data as necessary, and generates frequency distributions and identifies the rearranged DNA sequences for each target. Total of 198 peripheral blood samples were also tested by the capillary electrophoresis (CE) based IdentiClone® IGK assay.
Results
Data generated with the IGK NGS assay and bioinformatics software identified clonality and corresponding DNA sequences of IGK Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements. Excellent linearity of the IGK NGS assay was obtained from contrived cell line DNA in the range of 2.5 – 20% when running individually (R2=0.9897) or in combination with IGH and TRG assays (R2=0.9905). The concordance between the 198 samples tested by IGK NGS and CE assays was 77%. There were 43 samples indentified to be clonal out of 198 samples tested by both IGK and IGH NGS assays. Among those samples, 31 and 25 samples were clonal for IGK and IGH, respectively, and 18 samples were clonal for both IGK and IGH. It was demonstrated that the NGS assay allows for easy tracking of clonal populations at the sensitivity of 10-4 or at even lower limits of dectection, provided sufficient amount of DNA is tested.
Conclusion
A comprehensive assay has been developed for the MiSeq® platform that identifies clonal IGK rearrangements and the associated specific DNA sequences. The IGK assay, in combination with the IGH assay, increased the detection of clonality by 72% comparing to IGH alone. The assay can be used both to detect clonality and monitor the identified clonal populations in MRD.
Session topic: E-poster
Keyword(s): B-CLL, Clonality, Diagnosis, Non-Hodgkin's lymphoma
Type: Eposter Presentation
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. In addition to V-D-J rearrangement, the IGK Kde locus can rearrange to Vk gene and to an isolated RSS in the Jk-Ck intron (INTR).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. The clinical relevance of minimal residual disease (MRD) detection in lymphoproliferative disease has been demonstrated by many studies. However, allele-specific primers are required for the qPCR-based MRD monitoring.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 MRD monitoring without the requirement of allele-specific designs.
Aims
To establish an IGK NGS assay that, combined with an IGH NGS assay, will increase the chance of identifying clonal populations and provide easy tracking of identified clonal population for MRD in lymphoid malignancies.
Methods
Genomic DNA from cell line, peripheral blood, bone marrow aspirates, and FFPE were tested for IGK rearrangements using the LymphoTrack® IGK MiSeq® Assay (Invivoscribe Inc., San Diego, USA). The IGK libraries were run individually or in combination with IGH and or TRG libraries. The sequencing data was analyzed using LymphoTrack® bioinformatics software, which performs the separation of IGK, IGH, and TRG data as necessary, and generates frequency distributions and identifies the rearranged DNA sequences for each target. Total of 198 peripheral blood samples were also tested by the capillary electrophoresis (CE) based IdentiClone® IGK assay.
Results
Data generated with the IGK NGS assay and bioinformatics software identified clonality and corresponding DNA sequences of IGK Vk-Jk, Vk-Kde, and INTR-Kde gene rearrangements. Excellent linearity of the IGK NGS assay was obtained from contrived cell line DNA in the range of 2.5 – 20% when running individually (R2=0.9897) or in combination with IGH and TRG assays (R2=0.9905). The concordance between the 198 samples tested by IGK NGS and CE assays was 77%. There were 43 samples indentified to be clonal out of 198 samples tested by both IGK and IGH NGS assays. Among those samples, 31 and 25 samples were clonal for IGK and IGH, respectively, and 18 samples were clonal for both IGK and IGH. It was demonstrated that the NGS assay allows for easy tracking of clonal populations at the sensitivity of 10-4 or at even lower limits of dectection, provided sufficient amount of DNA is tested.
Conclusion
A comprehensive assay has been developed for the MiSeq® platform that identifies clonal IGK rearrangements and the associated specific DNA sequences. The IGK assay, in combination with the IGH assay, increased the detection of clonality by 72% comparing to IGH alone. The assay can be used both to detect clonality and monitor the identified clonal populations in MRD.
Session topic: E-poster
Keyword(s): B-CLL, Clonality, Diagnosis, Non-Hodgkin's lymphoma
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