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Clinically relevant variation in FLT3-ITD quantitation as a result of PCR cycle number and ITD insertion size
Ing S Tiong1, Nikky Andrieska1, Phuong Dang1
1Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Vic, Australia.
Abstract:
FLT3 internal tandem duplication (ITD) quantitation is key to prognostication in acute myeloid leukaemia (AML). One potential source of variability in the allelic ratio (AR) is the number of polymerase chain reaction (PCR) cycles used. Using 30 archived samples of varying ITD lengths and AR, we compared two FLT3-ITD assays (Huang and RATIFY), evaluated the effect of PCR cycle number on each assay, and examined the potential clinical consequences. Huang and RATIFY assays at 35 and 27 PCR cycles, respectively, were highly concordant. A progressive decrease in AR (median 47%) was observed with the RATIFY assay when the PCR cycles were increased from 27 to 35 cycles, potentially impacting risk categorisation in 29% of patients. In contrast, minimal change in AR was observed with the Huang assay. Hence, both FLT3-ITD assays were almost identical using respective standard conditions, but the effect of PCR cycle number is assay-dependent, which may impact risk stratification in AML.
Insights
Polymerase chain reaction (PCR) cycle number affects FLT3-internal tandem duplication (ITD) quantitation in acute myeloid leukemia (AML). The RATIFY assay showed significant allelic ratio variability with increased PCR cycles, unlike the Huang assay, impacting AML prognostication.
Area of Science:
- Hematology
- Molecular Biology
- Clinical Diagnostics
Background:
- FLT3 internal tandem duplication (ITD) quantitation is crucial for acute myeloid leukemia (AML) prognostication.
- Variability in allelic ratio (AR) measurements can arise from factors like polymerase chain reaction (PCR) cycle number.
- Standardized and reliable FLT3-ITD testing is essential for accurate patient risk stratification.
Purpose of the Study:
- To compare the performance of two FLT3-ITD assays (Huang and RATIFY).
- To evaluate the impact of varying PCR cycle numbers on AR quantitation for each assay.
- To assess the potential clinical consequences of assay-dependent variability in FLT3-ITD testing for AML patients.
Main Methods:
- Analysis of 30 archived AML samples with diverse FLT3-ITD lengths and ARs.
- Comparison of Huang and RATIFY FLT3-ITD assays under different PCR cycle conditions.
- Evaluation of the effect of increasing PCR cycles on AR measurements for both assays.
Main Results:
- Both Huang and RATIFY assays demonstrated high concordance at their respective standard PCR cycle numbers (35 and 27).
- Increasing PCR cycles from 27 to 35 in the RATIFY assay led to a median 47% decrease in AR, potentially affecting risk categorization in 29% of patients.
- The Huang assay exhibited minimal change in AR with increased PCR cycles, showing greater stability.
Conclusions:
- While both FLT3-ITD assays are comparable under standard conditions, their sensitivity to PCR cycle number varies significantly.
- Assay-dependent variability in FLT3-ITD quantitation due to PCR cycling can impact AML risk stratification.
- Careful consideration of PCR cycling protocols is necessary to ensure consistent and reliable FLT3-ITD testing for AML prognostication.
