BCR-ABL1 MONITORING ON THE IS USING AN ANALYTICALY AND CLINICALLY VALIDATED MULTIPLEX ASSAY DIRECTLY ALIGNED TO THE WHO PRIMARY STANDARDS
(Abstract release date: 05/19/16)
EHA Library. Brown J. 06/09/16; 132662; E1113
Disclosure(s): Employment by Asuragen, Inc.
Dr. Justin Brown
Contributions
Contributions
Abstract
Abstract: E1113
Type: Eposter Presentation
Background
Detection of BCR-ABL1 e13a2/e14a2 fusion transcripts (major breakpoint, M-BCR) of t(9;22) is important in studying tumor burden in CML. The International Scale (IS) was established to standardize the reporting of these transcripts against a common baseline. As newer TKI therapies create deeper responses, analytical sensitivity has become a critical topic in investigations into TKI discontinuation, where researchers require an assay that confidently calls molecular responses of ≥4.5 logs below baseline (0.0032%IS or MR4.5). This has led to various reporting formats as a patient achieves deep response over time, creating a non-contiguous language of monitoring: baseline, 10%IS, 1%IS, MMR, MR4, and MR4.5.
Aims
We describe the analytical and clinical validation of a multiplex assay system reporting continuous MR values via automated software analysis, clinical accuracy at MR3, analytical sensitivity of MR4.7, and direct traceability to the WHO Primary BCR-ABL1 reference materials without requiring establishment and revalidation of a conversion factor.
Methods
We developed reagents for RT-qPCR, both steps performed on the ABI 7500 Fast Dx. Armored RNA Quant® (ARQ) technology was employed to generate a blend of nuclease-resistant BCR-ABL1 and ABL1 RNA transcripts to calibrate and control the system. A single four-point standard curve using ARQ blends mimics the WHO Primary BCR-ABL1 reference materials and accounts for the relative batch run-specific efficiency of the RT step. cDNA generation and qPCR were optimized, including allowance of high mass of nucleic acid without inhibition. Residual clinical RNAs were tested to estimate LOD at minimum RNA input. Software was developed, including a floating, traceable logic algorithm to ensure that sufficient ABL1 was detected to protect this LOD. A multi‐center clinical outcome study was conducted at 3 clinical laboratories to validate clinical monitoring. Performance was assessed by event‐free survival (EFS) at 32‐40 months against test results at 12‐18 months on TKI as estimated by the Kaplan Meier survival function. A total of 139 samples from 98 patients were enrolled at 2 clinical sites.
Results
We surpassed the desired analytical sensitivity: 1680 measurements were generated across 28 levels and yielded an LOD estimate of 95% positivity by probit analysis of MR4.74 (0.0018%IS). LOQ was similar. Despite deep analytical sensitivity, this system maintains analytical specificity (non-leukemic and non-CML leukemic specimens were true negative). Linearity was observed from at least MR0.3 (50%IS) to MR4.7 (0.002%IS). Single-site precision included lot, instrument, operator, and run, and was verified as SD ≤0.13 for MR values ≤3.7. Multi-site precision included site, instrument, operator, and day, and was verified as SD ≤0.10 for MR values ≤3.7. Traceability to the higher order WHO standards was demonstrated. In the clinical study, the difference in EFS between
Conclusion
The BCR-ABL1 test improves workflow with its streamlined reagent formulation, multiplex assay format, and automated software analysis. It facilitates assessment on the IS without conversion (through integrated ARQ materials traceable to the WHO Primary), reports results on a continuous scale (as both MR and %IS values), and generates results sufficient for studies in deep molecular responses. Further, it is clinically validated to predict EFS outcomes at the MR3 level.
Session topic: E-poster
Keyword(s): BCR-ABL, Chronic myeloid leukemia, Clinical outcome, Monitor
Type: Eposter Presentation
Background
Detection of BCR-ABL1 e13a2/e14a2 fusion transcripts (major breakpoint, M-BCR) of t(9;22) is important in studying tumor burden in CML. The International Scale (IS) was established to standardize the reporting of these transcripts against a common baseline. As newer TKI therapies create deeper responses, analytical sensitivity has become a critical topic in investigations into TKI discontinuation, where researchers require an assay that confidently calls molecular responses of ≥4.5 logs below baseline (0.0032%IS or MR4.5). This has led to various reporting formats as a patient achieves deep response over time, creating a non-contiguous language of monitoring: baseline, 10%IS, 1%IS, MMR, MR4, and MR4.5.
Aims
We describe the analytical and clinical validation of a multiplex assay system reporting continuous MR values via automated software analysis, clinical accuracy at MR3, analytical sensitivity of MR4.7, and direct traceability to the WHO Primary BCR-ABL1 reference materials without requiring establishment and revalidation of a conversion factor.
Methods
We developed reagents for RT-qPCR, both steps performed on the ABI 7500 Fast Dx. Armored RNA Quant® (ARQ) technology was employed to generate a blend of nuclease-resistant BCR-ABL1 and ABL1 RNA transcripts to calibrate and control the system. A single four-point standard curve using ARQ blends mimics the WHO Primary BCR-ABL1 reference materials and accounts for the relative batch run-specific efficiency of the RT step. cDNA generation and qPCR were optimized, including allowance of high mass of nucleic acid without inhibition. Residual clinical RNAs were tested to estimate LOD at minimum RNA input. Software was developed, including a floating, traceable logic algorithm to ensure that sufficient ABL1 was detected to protect this LOD. A multi‐center clinical outcome study was conducted at 3 clinical laboratories to validate clinical monitoring. Performance was assessed by event‐free survival (EFS) at 32‐40 months against test results at 12‐18 months on TKI as estimated by the Kaplan Meier survival function. A total of 139 samples from 98 patients were enrolled at 2 clinical sites.
Results
We surpassed the desired analytical sensitivity: 1680 measurements were generated across 28 levels and yielded an LOD estimate of 95% positivity by probit analysis of MR4.74 (0.0018%IS). LOQ was similar. Despite deep analytical sensitivity, this system maintains analytical specificity (non-leukemic and non-CML leukemic specimens were true negative). Linearity was observed from at least MR0.3 (50%IS) to MR4.7 (0.002%IS). Single-site precision included lot, instrument, operator, and run, and was verified as SD ≤0.13 for MR values ≤3.7. Multi-site precision included site, instrument, operator, and day, and was verified as SD ≤0.10 for MR values ≤3.7. Traceability to the higher order WHO standards was demonstrated. In the clinical study, the difference in EFS between
Conclusion
The BCR-ABL1 test improves workflow with its streamlined reagent formulation, multiplex assay format, and automated software analysis. It facilitates assessment on the IS without conversion (through integrated ARQ materials traceable to the WHO Primary), reports results on a continuous scale (as both MR and %IS values), and generates results sufficient for studies in deep molecular responses. Further, it is clinically validated to predict EFS outcomes at the MR3 level.
Session topic: E-poster
Keyword(s): BCR-ABL, Chronic myeloid leukemia, Clinical outcome, Monitor
Abstract: E1113
Type: Eposter Presentation
Background
Detection of BCR-ABL1 e13a2/e14a2 fusion transcripts (major breakpoint, M-BCR) of t(9;22) is important in studying tumor burden in CML. The International Scale (IS) was established to standardize the reporting of these transcripts against a common baseline. As newer TKI therapies create deeper responses, analytical sensitivity has become a critical topic in investigations into TKI discontinuation, where researchers require an assay that confidently calls molecular responses of ≥4.5 logs below baseline (0.0032%IS or MR4.5). This has led to various reporting formats as a patient achieves deep response over time, creating a non-contiguous language of monitoring: baseline, 10%IS, 1%IS, MMR, MR4, and MR4.5.
Aims
We describe the analytical and clinical validation of a multiplex assay system reporting continuous MR values via automated software analysis, clinical accuracy at MR3, analytical sensitivity of MR4.7, and direct traceability to the WHO Primary BCR-ABL1 reference materials without requiring establishment and revalidation of a conversion factor.
Methods
We developed reagents for RT-qPCR, both steps performed on the ABI 7500 Fast Dx. Armored RNA Quant® (ARQ) technology was employed to generate a blend of nuclease-resistant BCR-ABL1 and ABL1 RNA transcripts to calibrate and control the system. A single four-point standard curve using ARQ blends mimics the WHO Primary BCR-ABL1 reference materials and accounts for the relative batch run-specific efficiency of the RT step. cDNA generation and qPCR were optimized, including allowance of high mass of nucleic acid without inhibition. Residual clinical RNAs were tested to estimate LOD at minimum RNA input. Software was developed, including a floating, traceable logic algorithm to ensure that sufficient ABL1 was detected to protect this LOD. A multi‐center clinical outcome study was conducted at 3 clinical laboratories to validate clinical monitoring. Performance was assessed by event‐free survival (EFS) at 32‐40 months against test results at 12‐18 months on TKI as estimated by the Kaplan Meier survival function. A total of 139 samples from 98 patients were enrolled at 2 clinical sites.
Results
We surpassed the desired analytical sensitivity: 1680 measurements were generated across 28 levels and yielded an LOD estimate of 95% positivity by probit analysis of MR4.74 (0.0018%IS). LOQ was similar. Despite deep analytical sensitivity, this system maintains analytical specificity (non-leukemic and non-CML leukemic specimens were true negative). Linearity was observed from at least MR0.3 (50%IS) to MR4.7 (0.002%IS). Single-site precision included lot, instrument, operator, and run, and was verified as SD ≤0.13 for MR values ≤3.7. Multi-site precision included site, instrument, operator, and day, and was verified as SD ≤0.10 for MR values ≤3.7. Traceability to the higher order WHO standards was demonstrated. In the clinical study, the difference in EFS between
Conclusion
The BCR-ABL1 test improves workflow with its streamlined reagent formulation, multiplex assay format, and automated software analysis. It facilitates assessment on the IS without conversion (through integrated ARQ materials traceable to the WHO Primary), reports results on a continuous scale (as both MR and %IS values), and generates results sufficient for studies in deep molecular responses. Further, it is clinically validated to predict EFS outcomes at the MR3 level.
Session topic: E-poster
Keyword(s): BCR-ABL, Chronic myeloid leukemia, Clinical outcome, Monitor
Type: Eposter Presentation
Background
Detection of BCR-ABL1 e13a2/e14a2 fusion transcripts (major breakpoint, M-BCR) of t(9;22) is important in studying tumor burden in CML. The International Scale (IS) was established to standardize the reporting of these transcripts against a common baseline. As newer TKI therapies create deeper responses, analytical sensitivity has become a critical topic in investigations into TKI discontinuation, where researchers require an assay that confidently calls molecular responses of ≥4.5 logs below baseline (0.0032%IS or MR4.5). This has led to various reporting formats as a patient achieves deep response over time, creating a non-contiguous language of monitoring: baseline, 10%IS, 1%IS, MMR, MR4, and MR4.5.
Aims
We describe the analytical and clinical validation of a multiplex assay system reporting continuous MR values via automated software analysis, clinical accuracy at MR3, analytical sensitivity of MR4.7, and direct traceability to the WHO Primary BCR-ABL1 reference materials without requiring establishment and revalidation of a conversion factor.
Methods
We developed reagents for RT-qPCR, both steps performed on the ABI 7500 Fast Dx. Armored RNA Quant® (ARQ) technology was employed to generate a blend of nuclease-resistant BCR-ABL1 and ABL1 RNA transcripts to calibrate and control the system. A single four-point standard curve using ARQ blends mimics the WHO Primary BCR-ABL1 reference materials and accounts for the relative batch run-specific efficiency of the RT step. cDNA generation and qPCR were optimized, including allowance of high mass of nucleic acid without inhibition. Residual clinical RNAs were tested to estimate LOD at minimum RNA input. Software was developed, including a floating, traceable logic algorithm to ensure that sufficient ABL1 was detected to protect this LOD. A multi‐center clinical outcome study was conducted at 3 clinical laboratories to validate clinical monitoring. Performance was assessed by event‐free survival (EFS) at 32‐40 months against test results at 12‐18 months on TKI as estimated by the Kaplan Meier survival function. A total of 139 samples from 98 patients were enrolled at 2 clinical sites.
Results
We surpassed the desired analytical sensitivity: 1680 measurements were generated across 28 levels and yielded an LOD estimate of 95% positivity by probit analysis of MR4.74 (0.0018%IS). LOQ was similar. Despite deep analytical sensitivity, this system maintains analytical specificity (non-leukemic and non-CML leukemic specimens were true negative). Linearity was observed from at least MR0.3 (50%IS) to MR4.7 (0.002%IS). Single-site precision included lot, instrument, operator, and run, and was verified as SD ≤0.13 for MR values ≤3.7. Multi-site precision included site, instrument, operator, and day, and was verified as SD ≤0.10 for MR values ≤3.7. Traceability to the higher order WHO standards was demonstrated. In the clinical study, the difference in EFS between
Conclusion
The BCR-ABL1 test improves workflow with its streamlined reagent formulation, multiplex assay format, and automated software analysis. It facilitates assessment on the IS without conversion (through integrated ARQ materials traceable to the WHO Primary), reports results on a continuous scale (as both MR and %IS values), and generates results sufficient for studies in deep molecular responses. Further, it is clinically validated to predict EFS outcomes at the MR3 level.
Session topic: E-poster
Keyword(s): BCR-ABL, Chronic myeloid leukemia, Clinical outcome, Monitor
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