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. 2025 Mar 1;17(5):854.
doi: 10.3390/cancers17050854.

Conventional PCR Versus Next Generation Sequencing for Diagnosis of FLT3, IDH and NPM1 Mutations in Acute Myeloid Leukemia: Results of the PETHEMA PCR-LMA Study

Blanca Boluda  1 , Rebeca Rodriguez-Veiga  1 , Claudia Sargas  2 , Rosa Ayala  3 , María J Larráyoz  4 , María Carmen Chillón  5 , Elena Soria-Saldise  6 , Cristina Bilbao  7 , Esther Prados de la Torre  8 , Irene Navarro  1 , David Martinez-Cuadron  1   9 , Cristina Gil  10 , Teresa Bernal  11 , Juan Bergua  12 , Lorenzo Algarra  13 , Mar Tormo  14 , Pilar Martínez-Sanchez  3 , Estrella Carrillo-Cruz  6 , Josefina Serrano  8 , Juan M Alonso-Domínguez  15 , Raimundo García  16 , Maria Luz Amigo  17 , Pilar Herrera-Puente  18 , María J Sayas  19 , Esperanza Lavilla-Rubira  20 , María José García-Pérez  21 , Julia Morán  22 , Esther Pérez-Santaolalla  23 , Natalia Alonso-Vence  24 , Ana Oliva  25 , Juan Antonio López  26 , Manuel Barrios  27 , María García-Fortes  28 , María Teresa Olave  29 , Jorge Labrador  30 , Joaquín Martínez-López  3 , María J Calasanz  4 , Ramón García-Sanz  5 , José A Pérez-Simón  6 , María T Gómez-Casares  7 , Joaquín Sánchez-Garcia  8 , Yolanda Mendizabal  1 , Eva Barragán  2   9 , Pau Montesinos  1   9 ; PETHEMA Group
Affiliations

Conventional PCR Versus Next Generation Sequencing for Diagnosis of FLT3, IDH and NPM1 Mutations in Acute Myeloid Leukemia: Results of the PETHEMA PCR-LMA Study

Blanca Boluda et al. Cancers (Basel). .

Abstract

Background/objectives: This PETHEMA PCR-LMA study aimed to evaluate whether mutations detected by NGS (VAF cut-off of ≥5%) correlate with NPM1, FLT3-ITD, FLT3-TKD, IDH1, and IDH2 mutations detected using conventional PCR (analytical sensitivity 3%) in a nationwide network of seven reference laboratories.

Methods: Between 2019 and 2021, 1685 adult AML patients with at least one centralized sample (NGS or PCR) at primary diagnosis or relapse/refractory episode were included.

Results: During this period, 1288 paired NGS/PCR samples (1094 at diagnosis, 103 at relapse and 88 at refractoriness) were analyzed. Considering PCR the gold-standard, for NPM1 NGS sensitivity was 98.5% and specificity 98.9%, for FLT3-ITD 73.8% and 99.6%, for FLT3-TKD 84.5% and 99.3%, for IDH1 98.7% and 98.7%, and for IDH2 99.1% and 97.7%, respectively. Overall concordance rate of positive results between NGS (and PCR was 95% (262/276) for NPM1, 72% (149/206) for FLT3-ITD, 74% (49/66) for FLT3-TKD, 87% (77/89) for IDH1 and 84% (107/127) for IDH2. Overall, median days from sample reception until report were 7 for PCR and 28 for NGS.

Conclusions: This study shows high concordance between NPM1 and IDH results using PCR and NGS. However, sensible important discrepancies are observed for FLT3 mutations. In our context, rapid screening for these druggable mutations should be performed by conventional PCR.

Keywords: NGS (next generation sequencing); PCR; acute myeloid leukemia.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Frequency of mutations at diagnosis by NGS.
Figure 2
Figure 2
Positive results by conventional PCR and NGS (VAF cut-off ≥5%).
Figure 3
Figure 3
Sensitivity and specificity of NGS (VAF cut-off ≥5%) with respect to PCR.
Figure 4
Figure 4
Concordance of positive results between PCR and NGS (VAF cut-off ≥5%).
Figure 5
Figure 5
Concordance of positive results with PCR and NGS (VAF cut-off ≥1%).
See this image and copyright information in PMC

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