CLONE-SPECIFIC IGH PLASMIDS AS AN ALTERNATIVE STANDARD FOR ASSESSMENT OF MINIMAL RESIDUAL DISEASE BY REAL-TIME QUANTITATIVE PCR IN FOLLICULAR LYMPHOMA
(Abstract release date: 05/19/16)
EHA Library. Laubenstein T. 06/09/16; 132710; E1161
Disclosure(s): My Congress fee as well travelling costs were paid/ sponsored by JAZZ Pharmaceuticals
Mr. Tobias Laubenstein
Contributions
Contributions
Abstract
Abstract: E1161
Type: Eposter Presentation
Background
Development of novel treatment options, led to improved clinical outcome in follicular lymphoma (FL). Additionally sensitive diagnostic tools have been established, able to detect low amounts of malignant cells, so-called minimal residual disease (MRD). Real-time quantitative pcr (RQ-PCR) using the t(14;18) as a molecular target is beeing broadly applied in FL. In cases with no PCR-detectable t(14;18), clonal rearrangements of the immunoglobulin genes (IGH) can be used, limited by the required information of lymphoma infiltration in the diagnostic sample, which is not regularly available due to nodal disease character. Cloned IGH-plasmids as an external standard for RQ-PCR could be a useful alternative.
Aims
This study investigated, whether RQ-PCR using clone-specific IGH-plasmids as standards for quantitative MRD-assessment in FL, is an accurate, reproducible and feasible alternative if no t(14;18) is available as a PCR-target.
Methods
IGH-rearrangements of a b-cell-lymphoma cell-line and 24 FL patients, amplified by FR1-IGH-PCR, were sequenced for identification of the clone-specific VDJ-region. Allel specific oligonucleotides were designed to the CDR3- and the FR3-region as reverse/ forward primers for RQ-PCR. Antisense consensus probes fitting to the FR3-region were used if possible, otherwise ASO-probes were designed. The IGH-rearrangements were cloned into plasmids and serial dilutions were performed for the cell-line and each patient to serve as external standards for RQ-PCR. Initially a serial dilution of cell-line DNA containing known IGH-copies was quantified by the plasmid-standard. Subsequently amplification characteristics of the IGH-plasmids of 24 FL patients were compared to each other and to relative genomic DNA (gDNA)-standards. Finally MRD-levels of follow-up samples, determined by using IGH-plasmid- and t(14;18)-cell-line- or IGH-gDNA-standards were compared.
Results
IGH-copies of a cell-line sample could be quantified accurately using IGH-plasmid-standards (median deviation: 6,9%, SD: 3,9%, corr. coefficient: 0,999). IGH-plasmid-standard-curves of the 24 patients indicated high conformity referring to slope (median: -3,39; SD: 0,08), correllation coefficient (median: 0,998; SD: 0,001), efficacy (median: 0,97; SD: 0,03), sensitivity (median: 10-5) and specificity (median: 5x10-5). Compared to gDNA- plasmid-standards showed comparable sensitivity (median: 10-5 vs. 1,25*x10-5; SD: 1,2x10-5 vs. 2,69x10-5) and a superior specificity (median: 5x10-5 vs. 1,41x10-4; SD: 2,5x10-5 vs. 2,19x10-4). Moreover a significant superiority of the plasmid-standards was seen regarding to slope (median: -3,39 vs. -3,67; SD: 0,08 vs. 0,18; p: 0,008), correlation coefficient (median: 0,998 vs. 0,990; SD: 0,001 vs. 0,011; p: 0,009), and efficiency (median: 0,97 vs. 0,87; SD: 0,03 vs. 0,06; p: 0,008). In a final step patient follow-up samples were quantified by different strategies, using clone-specific IGH-plasmids vs. IGH-gDNA or t(14;18)-cell-line-DNA respectively (total of 126 comparisons). In 92/126 (73,0%) of these comparisons Δmrd-level was < 0,5 log-steps, in 21/126 (16,7%) within 0,5-1 log-steps, in 4/126 (3,2%) within 1-2 log-steps. Only in 9/126 (7,2%) cases Δmrd-level was > 2 log-steps.
Conclusion
Quantitative MRD-assessment in FL using clone-/ patient-specific IGH-plasmids as external standards for RQ-PCR, represents an accurate, reproducible and feasible strategy and can therefore be used in cases with no pcr-detectable t(14;18).
Session topic: E-poster
Keyword(s): Follicular lymphoma, IgH rearrangment, MRD, RQ-PCR
Type: Eposter Presentation
Background
Development of novel treatment options, led to improved clinical outcome in follicular lymphoma (FL). Additionally sensitive diagnostic tools have been established, able to detect low amounts of malignant cells, so-called minimal residual disease (MRD). Real-time quantitative pcr (RQ-PCR) using the t(14;18) as a molecular target is beeing broadly applied in FL. In cases with no PCR-detectable t(14;18), clonal rearrangements of the immunoglobulin genes (IGH) can be used, limited by the required information of lymphoma infiltration in the diagnostic sample, which is not regularly available due to nodal disease character. Cloned IGH-plasmids as an external standard for RQ-PCR could be a useful alternative.
Aims
This study investigated, whether RQ-PCR using clone-specific IGH-plasmids as standards for quantitative MRD-assessment in FL, is an accurate, reproducible and feasible alternative if no t(14;18) is available as a PCR-target.
Methods
IGH-rearrangements of a b-cell-lymphoma cell-line and 24 FL patients, amplified by FR1-IGH-PCR, were sequenced for identification of the clone-specific VDJ-region. Allel specific oligonucleotides were designed to the CDR3- and the FR3-region as reverse/ forward primers for RQ-PCR. Antisense consensus probes fitting to the FR3-region were used if possible, otherwise ASO-probes were designed. The IGH-rearrangements were cloned into plasmids and serial dilutions were performed for the cell-line and each patient to serve as external standards for RQ-PCR. Initially a serial dilution of cell-line DNA containing known IGH-copies was quantified by the plasmid-standard. Subsequently amplification characteristics of the IGH-plasmids of 24 FL patients were compared to each other and to relative genomic DNA (gDNA)-standards. Finally MRD-levels of follow-up samples, determined by using IGH-plasmid- and t(14;18)-cell-line- or IGH-gDNA-standards were compared.
Results
IGH-copies of a cell-line sample could be quantified accurately using IGH-plasmid-standards (median deviation: 6,9%, SD: 3,9%, corr. coefficient: 0,999). IGH-plasmid-standard-curves of the 24 patients indicated high conformity referring to slope (median: -3,39; SD: 0,08), correllation coefficient (median: 0,998; SD: 0,001), efficacy (median: 0,97; SD: 0,03), sensitivity (median: 10-5) and specificity (median: 5x10-5). Compared to gDNA- plasmid-standards showed comparable sensitivity (median: 10-5 vs. 1,25*x10-5; SD: 1,2x10-5 vs. 2,69x10-5) and a superior specificity (median: 5x10-5 vs. 1,41x10-4; SD: 2,5x10-5 vs. 2,19x10-4). Moreover a significant superiority of the plasmid-standards was seen regarding to slope (median: -3,39 vs. -3,67; SD: 0,08 vs. 0,18; p: 0,008), correlation coefficient (median: 0,998 vs. 0,990; SD: 0,001 vs. 0,011; p: 0,009), and efficiency (median: 0,97 vs. 0,87; SD: 0,03 vs. 0,06; p: 0,008). In a final step patient follow-up samples were quantified by different strategies, using clone-specific IGH-plasmids vs. IGH-gDNA or t(14;18)-cell-line-DNA respectively (total of 126 comparisons). In 92/126 (73,0%) of these comparisons Δmrd-level was < 0,5 log-steps, in 21/126 (16,7%) within 0,5-1 log-steps, in 4/126 (3,2%) within 1-2 log-steps. Only in 9/126 (7,2%) cases Δmrd-level was > 2 log-steps.
Conclusion
Quantitative MRD-assessment in FL using clone-/ patient-specific IGH-plasmids as external standards for RQ-PCR, represents an accurate, reproducible and feasible strategy and can therefore be used in cases with no pcr-detectable t(14;18).
Session topic: E-poster
Keyword(s): Follicular lymphoma, IgH rearrangment, MRD, RQ-PCR
Abstract: E1161
Type: Eposter Presentation
Background
Development of novel treatment options, led to improved clinical outcome in follicular lymphoma (FL). Additionally sensitive diagnostic tools have been established, able to detect low amounts of malignant cells, so-called minimal residual disease (MRD). Real-time quantitative pcr (RQ-PCR) using the t(14;18) as a molecular target is beeing broadly applied in FL. In cases with no PCR-detectable t(14;18), clonal rearrangements of the immunoglobulin genes (IGH) can be used, limited by the required information of lymphoma infiltration in the diagnostic sample, which is not regularly available due to nodal disease character. Cloned IGH-plasmids as an external standard for RQ-PCR could be a useful alternative.
Aims
This study investigated, whether RQ-PCR using clone-specific IGH-plasmids as standards for quantitative MRD-assessment in FL, is an accurate, reproducible and feasible alternative if no t(14;18) is available as a PCR-target.
Methods
IGH-rearrangements of a b-cell-lymphoma cell-line and 24 FL patients, amplified by FR1-IGH-PCR, were sequenced for identification of the clone-specific VDJ-region. Allel specific oligonucleotides were designed to the CDR3- and the FR3-region as reverse/ forward primers for RQ-PCR. Antisense consensus probes fitting to the FR3-region were used if possible, otherwise ASO-probes were designed. The IGH-rearrangements were cloned into plasmids and serial dilutions were performed for the cell-line and each patient to serve as external standards for RQ-PCR. Initially a serial dilution of cell-line DNA containing known IGH-copies was quantified by the plasmid-standard. Subsequently amplification characteristics of the IGH-plasmids of 24 FL patients were compared to each other and to relative genomic DNA (gDNA)-standards. Finally MRD-levels of follow-up samples, determined by using IGH-plasmid- and t(14;18)-cell-line- or IGH-gDNA-standards were compared.
Results
IGH-copies of a cell-line sample could be quantified accurately using IGH-plasmid-standards (median deviation: 6,9%, SD: 3,9%, corr. coefficient: 0,999). IGH-plasmid-standard-curves of the 24 patients indicated high conformity referring to slope (median: -3,39; SD: 0,08), correllation coefficient (median: 0,998; SD: 0,001), efficacy (median: 0,97; SD: 0,03), sensitivity (median: 10-5) and specificity (median: 5x10-5). Compared to gDNA- plasmid-standards showed comparable sensitivity (median: 10-5 vs. 1,25*x10-5; SD: 1,2x10-5 vs. 2,69x10-5) and a superior specificity (median: 5x10-5 vs. 1,41x10-4; SD: 2,5x10-5 vs. 2,19x10-4). Moreover a significant superiority of the plasmid-standards was seen regarding to slope (median: -3,39 vs. -3,67; SD: 0,08 vs. 0,18; p: 0,008), correlation coefficient (median: 0,998 vs. 0,990; SD: 0,001 vs. 0,011; p: 0,009), and efficiency (median: 0,97 vs. 0,87; SD: 0,03 vs. 0,06; p: 0,008). In a final step patient follow-up samples were quantified by different strategies, using clone-specific IGH-plasmids vs. IGH-gDNA or t(14;18)-cell-line-DNA respectively (total of 126 comparisons). In 92/126 (73,0%) of these comparisons Δmrd-level was < 0,5 log-steps, in 21/126 (16,7%) within 0,5-1 log-steps, in 4/126 (3,2%) within 1-2 log-steps. Only in 9/126 (7,2%) cases Δmrd-level was > 2 log-steps.
Conclusion
Quantitative MRD-assessment in FL using clone-/ patient-specific IGH-plasmids as external standards for RQ-PCR, represents an accurate, reproducible and feasible strategy and can therefore be used in cases with no pcr-detectable t(14;18).
Session topic: E-poster
Keyword(s): Follicular lymphoma, IgH rearrangment, MRD, RQ-PCR
Type: Eposter Presentation
Background
Development of novel treatment options, led to improved clinical outcome in follicular lymphoma (FL). Additionally sensitive diagnostic tools have been established, able to detect low amounts of malignant cells, so-called minimal residual disease (MRD). Real-time quantitative pcr (RQ-PCR) using the t(14;18) as a molecular target is beeing broadly applied in FL. In cases with no PCR-detectable t(14;18), clonal rearrangements of the immunoglobulin genes (IGH) can be used, limited by the required information of lymphoma infiltration in the diagnostic sample, which is not regularly available due to nodal disease character. Cloned IGH-plasmids as an external standard for RQ-PCR could be a useful alternative.
Aims
This study investigated, whether RQ-PCR using clone-specific IGH-plasmids as standards for quantitative MRD-assessment in FL, is an accurate, reproducible and feasible alternative if no t(14;18) is available as a PCR-target.
Methods
IGH-rearrangements of a b-cell-lymphoma cell-line and 24 FL patients, amplified by FR1-IGH-PCR, were sequenced for identification of the clone-specific VDJ-region. Allel specific oligonucleotides were designed to the CDR3- and the FR3-region as reverse/ forward primers for RQ-PCR. Antisense consensus probes fitting to the FR3-region were used if possible, otherwise ASO-probes were designed. The IGH-rearrangements were cloned into plasmids and serial dilutions were performed for the cell-line and each patient to serve as external standards for RQ-PCR. Initially a serial dilution of cell-line DNA containing known IGH-copies was quantified by the plasmid-standard. Subsequently amplification characteristics of the IGH-plasmids of 24 FL patients were compared to each other and to relative genomic DNA (gDNA)-standards. Finally MRD-levels of follow-up samples, determined by using IGH-plasmid- and t(14;18)-cell-line- or IGH-gDNA-standards were compared.
Results
IGH-copies of a cell-line sample could be quantified accurately using IGH-plasmid-standards (median deviation: 6,9%, SD: 3,9%, corr. coefficient: 0,999). IGH-plasmid-standard-curves of the 24 patients indicated high conformity referring to slope (median: -3,39; SD: 0,08), correllation coefficient (median: 0,998; SD: 0,001), efficacy (median: 0,97; SD: 0,03), sensitivity (median: 10-5) and specificity (median: 5x10-5). Compared to gDNA- plasmid-standards showed comparable sensitivity (median: 10-5 vs. 1,25*x10-5; SD: 1,2x10-5 vs. 2,69x10-5) and a superior specificity (median: 5x10-5 vs. 1,41x10-4; SD: 2,5x10-5 vs. 2,19x10-4). Moreover a significant superiority of the plasmid-standards was seen regarding to slope (median: -3,39 vs. -3,67; SD: 0,08 vs. 0,18; p: 0,008), correlation coefficient (median: 0,998 vs. 0,990; SD: 0,001 vs. 0,011; p: 0,009), and efficiency (median: 0,97 vs. 0,87; SD: 0,03 vs. 0,06; p: 0,008). In a final step patient follow-up samples were quantified by different strategies, using clone-specific IGH-plasmids vs. IGH-gDNA or t(14;18)-cell-line-DNA respectively (total of 126 comparisons). In 92/126 (73,0%) of these comparisons Δmrd-level was < 0,5 log-steps, in 21/126 (16,7%) within 0,5-1 log-steps, in 4/126 (3,2%) within 1-2 log-steps. Only in 9/126 (7,2%) cases Δmrd-level was > 2 log-steps.
Conclusion
Quantitative MRD-assessment in FL using clone-/ patient-specific IGH-plasmids as external standards for RQ-PCR, represents an accurate, reproducible and feasible strategy and can therefore be used in cases with no pcr-detectable t(14;18).
Session topic: E-poster
Keyword(s): Follicular lymphoma, IgH rearrangment, MRD, RQ-PCR
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