Performance of Amplicon-based Next Generation Sequencing in Detection and Quantification of FLT3-ITDs as Compared to Conventional PCR-based Testing
Cancer Genetics and Therapeutics
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Primary Categories:
- Cancer
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Secondary Categories:
- Cancer
Introduction:
FLT3 internal tandem duplications (ITDs) are biomarkers with important prognostic and therapeutic implications in patients with acute myeloid leukemia. Conventional methods for assessing FLT3-ITD status in clinical samples utilize PCR fragment analysis. Amplicon based NGS has been thought to have intrinsic difficulties in detecting large ITD events due to its limited read length. This study was conducted to compare the detection and quantification of FLT3-ITDs in clinical samples by conventional PCR fragment analysis and Amplicon-based NGS.
Methods:
DNA was extracted from 293 samples including bone marrow (151), peripheral blood (141) or cerebral spinal fluid (1) using Qiagen Mini Kit on QIACube. For conventional PCR fragment analysis, extracted DNA was PCR amplified with primers covering a 329bp region in exon 14 of FLT3 gene and PCR products were analyzed by capillary electrophoresis on ABI 3500XL (Thermo Fisher). An ITD in FLT3 is identified as any peak greater than the wild-type 329 bp one. For NGS, extracted DNA was loaded onto GenexusTM Integrated Sequencer with OncomineTM Myeloid Assay GX v2 (Thermo Fisher) following manufacture’s instruction. The sequencing data was analyzed by both manual curation and the default Oncomime extended 5.16 filters.
Results:
Among 293 clinical samples, 19 were positive for insertions in FLT3 gene by PCR fragment analysis. The ITDs ranged in size from 15 bp to 186 bp (25 ± 36 bp). Five specimens harbored two peaks corresponding to ITDs of different lengths. NGS detected 18 of these FLT-ITD variants with variant allele frequency ranging from 0.7% to 89%. The lone exception was a186-bp duplication in two specimens from the same patients collected at different stages of diseases. The overall sensitivity of NGS was 89.5%. In addition to multiple FLT3-ITDs in the same specimen, NGS detected FLT3-TKD variant in one of the samples. Among the 274 specimens negative for FLT3-ITDs by PCR, 273 were confirmed as negative by NGS. In a lone case, the NGS detected a complex indel event resulting in net deletion of 6 bp that was undetected by PCR, revealing specificity of 99.6%. Among all the FLT3-ITD variants detected by both methods, a linear correlation was observed regarding the duplication size, with slope of 1.020 and R2 of 0.999, and the overall accuracy of FLT3-ITDs in NGS was determined to be 99.0%, indicating high concordance level. Moreover, while the size of ITDs detected by PCR can vary by ±1 bp due to the intrinsic limitations of capillary electrophoresis, which may result in insertions being misinterpreted as out-of-frame (11 out of 25 ITDs), the NGS method accurately detected these insertion sequences and determined them to be in-frame insertions.
Conclusion:
In summary, the amplicon based NGS test detected FLT3-ITDs with high overall accuracy, high specificity, and good sensitivity. The NGS test accurately confirmed insertions as in-frame by providing precise insertion sequences and could also detect FLT3-TKD mutations, adding critical clinical information to the patient’s diagnosis. Taken together, our results demonstrated that FLT3-ITDs can be reliably detected by this amplicon based NGS assay with current default bioinformatic pipeline, although PCR fragment analysis remains superior in identifying very long FLT3-ITDs in initial diagnosis.
FLT3 internal tandem duplications (ITDs) are biomarkers with important prognostic and therapeutic implications in patients with acute myeloid leukemia. Conventional methods for assessing FLT3-ITD status in clinical samples utilize PCR fragment analysis. Amplicon based NGS has been thought to have intrinsic difficulties in detecting large ITD events due to its limited read length. This study was conducted to compare the detection and quantification of FLT3-ITDs in clinical samples by conventional PCR fragment analysis and Amplicon-based NGS.
Methods:
DNA was extracted from 293 samples including bone marrow (151), peripheral blood (141) or cerebral spinal fluid (1) using Qiagen Mini Kit on QIACube. For conventional PCR fragment analysis, extracted DNA was PCR amplified with primers covering a 329bp region in exon 14 of FLT3 gene and PCR products were analyzed by capillary electrophoresis on ABI 3500XL (Thermo Fisher). An ITD in FLT3 is identified as any peak greater than the wild-type 329 bp one. For NGS, extracted DNA was loaded onto GenexusTM Integrated Sequencer with OncomineTM Myeloid Assay GX v2 (Thermo Fisher) following manufacture’s instruction. The sequencing data was analyzed by both manual curation and the default Oncomime extended 5.16 filters.
Results:
Among 293 clinical samples, 19 were positive for insertions in FLT3 gene by PCR fragment analysis. The ITDs ranged in size from 15 bp to 186 bp (25 ± 36 bp). Five specimens harbored two peaks corresponding to ITDs of different lengths. NGS detected 18 of these FLT-ITD variants with variant allele frequency ranging from 0.7% to 89%. The lone exception was a186-bp duplication in two specimens from the same patients collected at different stages of diseases. The overall sensitivity of NGS was 89.5%. In addition to multiple FLT3-ITDs in the same specimen, NGS detected FLT3-TKD variant in one of the samples. Among the 274 specimens negative for FLT3-ITDs by PCR, 273 were confirmed as negative by NGS. In a lone case, the NGS detected a complex indel event resulting in net deletion of 6 bp that was undetected by PCR, revealing specificity of 99.6%. Among all the FLT3-ITD variants detected by both methods, a linear correlation was observed regarding the duplication size, with slope of 1.020 and R2 of 0.999, and the overall accuracy of FLT3-ITDs in NGS was determined to be 99.0%, indicating high concordance level. Moreover, while the size of ITDs detected by PCR can vary by ±1 bp due to the intrinsic limitations of capillary electrophoresis, which may result in insertions being misinterpreted as out-of-frame (11 out of 25 ITDs), the NGS method accurately detected these insertion sequences and determined them to be in-frame insertions.
Conclusion:
In summary, the amplicon based NGS test detected FLT3-ITDs with high overall accuracy, high specificity, and good sensitivity. The NGS test accurately confirmed insertions as in-frame by providing precise insertion sequences and could also detect FLT3-TKD mutations, adding critical clinical information to the patient’s diagnosis. Taken together, our results demonstrated that FLT3-ITDs can be reliably detected by this amplicon based NGS assay with current default bioinformatic pipeline, although PCR fragment analysis remains superior in identifying very long FLT3-ITDs in initial diagnosis.
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