Verifying Cost Effective Conventional PCR as an Alternative to NGS in Detecting FLT3-ITD in AML at Diagnostic Time Point – A Cross Sectional Study From a Tertiary Care Centre in North India

Background: Nucleophosmin1 (NPM1) and FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) are two important mutations for risk stratification in acute myeloid leukemia (AML). Newer molecular techniques, like targeted next-generation sequencing (NGS) have better sensitivity and ability to detect multiple mutations in a single run. However, their application is limited in resource-constrained set-ups. We aimed to compare the analytical performance of conventional PCR with NGS for detection of FLT3-ITD mutations at diagnosis and to analyze the prevalence of NPM1 and FLT3-ITD mutations in AML in this region.Methods: Conventional PCR and real-time PCR were performed in 130 AMLs to detect FLT3-ITD and NPM1 mutations (A, B or D mutations) respectively. Sanger sequencing was performed in 21 NPM1 mutated samples for sub-typing. Targeted NGS (Ion Torrent platform) was performed in 20 random paired samples to detect FLT3-ITD mutation for comparison of analytic performance between these techniques. Result: Concordance between conventional PCR and targeted NGS was high (95%) with a good agreement (kappa ratio=0.8). Fourteen (10.8%) cases had both the mutations, 12 (9.2%) and 27 (20.8%) cases had NPM1 mutation and FLT3-ITD mutation respectively. NPM1 mutant type A was detected in 90.5% (19/21) and type D in 9.5% (2/21) patients.Conclusion: Conventional PCR was compared well with targeted NGS (Ion Torrent platform) in detecting FLT3-ITD mutation. This finding supports a complementary role for PCR alongside NGS for FLT3-ITD detection in diagnostic samples specially in LMIC. Compared to published data, frequency of isolated NPM1 mutation was lower while that of FLT3-ITD mutation was higher in our AML cohort.