Prognostic impact of variant allele frequency in intensively treated patients with NPM1-mutated AML: a PETHEMA study

NPM1-mutated acute myeloid leukemia (AML) is genetically well-defined, but clinical outcomes remain heterogeneous, suggesting that quantitative clonal features may refine current risk stratification. We analyzed 688 intensively treated NPM1-mutated AML integrating variant allele frequency (VAF), mutation order, and clonal architecture inferred by PyClone and ClonEvol. Co-mutations were present in 97% of patients (median = 3 per case), dominated by DNMT3A (49%), FLT3-ITD (46%), TET2 (22%), and IDH2 (20%). Prognostic modelling of NPM1 VAF identified an optimal cut-off of 31.44%, defining NPM1high and NPM1low groups. NPM1low correlated with splicing-related alterations and independently predicted inferior overall (HR = 1.46; p = 0.037) and relapse-free survival (HR = 1.40; p = 0.036). Gene-specific VAF analyses revealed divergent effects across partners, high DNMT3A, FLT3-OTHER, KRAS, and PTPN11 burdens were adverse, whereas high IDH2 VAF was protective. Combined models showed that patients with NPM1high and favorable co-mutation VAFs had the best outcomes, while dual unfavorable burdens conferred the poorest survival. Mutation ordering inferred from VAFs positioned NPM1 after epigenetic and splicing lesions but before signaling and transcription-factor mutations. Non-canonical orders, such as early FLT3-OTHER/TKD or WT1 prior to NPM1, significantly stratified outcomes. Clonal reconstruction revealed predominantly linear evolutionary trajectories (84.3%), with increased mutational burden and clonal diversity associating with inferior survival. Notably, intra-clonal co-localization of NPM1 with IDH1 or TET2 was associated with improved outcomes, whereas co-localization with WT1 predicted dismal prognosis. These results demonstrate that quantitative and structural dimensions of clonality refine the biological and prognostic landscape of NPM1-mutated AML beyond mutational status alone.

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Journal
Blood
Published
2026-06-08
DOI
https://doi.org/10.1182/blood.2026033576
Primary Topic
Acute Myeloid Leukemia Research
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article
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article

Prognostic impact of variant allele frequency in intensively treated patients with NPM1-mutated AML: a PETHEMA study

Victor Noriega, Carmen Martínez Chamorro, R. Colmenares, Elena Soria et al.
Blood
Acute Myeloid Leukemia Research
article

Prognostic impact of variant allele frequency in intensively treated patients with NPM1-mutated AML: a PETHEMA study

Victor Noriega, Carmen Martínez Chamorro, R. Colmenares, Elena Soria, Lourdes Hermosín, Claudia Sargas, Juan Miguel Bergua Burgués, Isabel Cano‐Ferri, Norma C. Gutiérrez, Raimundo García‐Boyero, Jesús‐Lorenzo Algarra, María Solé‐Rodríguez, María‐Luz Amigo, María Teresa Gómez‐Casares, María del Carmen García Garay, María García‐Fortes, Josè Antonio Pérez-Simón, M.J. García Pérez, Ana Oliva, Esther Pérez Santaolalla, Cristina Bilbao‐Sieyro, Eduardo Rodríguez-Arbolí, Maria J José Calasanz, Joaquín Sánchez-García, Teresa Bernal del Castillo, Maria C. Mateos, Antonio Solana-Altabella, Irene Navarro, Cristina Gil, José Vicente Gil, Francisco Ibañez-Alis, Carlos Rodríguez-Medina, Mayte T Olave, Joaquín Martínez-López, Esther Prados de la Torre, Eva Barragan, María José Larrayoz, Beatriz Martín-Herreros, Mercedes Colorado, Rosa Ayala, Juan Manuel Alonso-Domínguez, Agata Almela-Gallego, Mar Tormo, Josefina Serrano, Pau Montesinos, María del Carmen Couto, M. Carmen Chillon, Dolores Dolores Madrigal-Toscano
article en

Abstract

NPM1-mutated acute myeloid leukemia (AML) is genetically well-defined, but clinical outcomes remain heterogeneous, suggesting that quantitative clonal features may refine current risk stratification. We analyzed 688 intensively treated NPM1-mutated AML integrating variant allele frequency (VAF), mutation order, and clonal architecture inferred by PyClone and ClonEvol. Co-mutations were present in 97% of patients (median = 3 per case), dominated by DNMT3A (49%), FLT3-ITD (46%), TET2 (22%), and IDH2 (20%). Prognostic modelling of NPM1 VAF identified an optimal cut-off of 31.44%, defining NPM1high and NPM1low groups. NPM1low correlated with splicing-related alterations and independently predicted inferior overall (HR = 1.46; p = 0.037) and relapse-free survival (HR = 1.40; p = 0.036). Gene-specific VAF analyses revealed divergent effects across partners, high DNMT3A, FLT3-OTHER, KRAS, and PTPN11 burdens were adverse, whereas high IDH2 VAF was protective. Combined models showed that patients with NPM1high and favorable co-mutation VAFs had the best outcomes, while dual unfavorable burdens conferred the poorest survival. Mutation ordering inferred from VAFs positioned NPM1 after epigenetic and splicing lesions but before signaling and transcription-factor mutations. Non-canonical orders, such as early FLT3-OTHER/TKD or WT1 prior to NPM1, significantly stratified outcomes. Clonal reconstruction revealed predominantly linear evolutionary trajectories (84.3%), with increased mutational burden and clonal diversity associating with inferior survival. Notably, intra-clonal co-localization of NPM1 with IDH1 or TET2 was associated with improved outcomes, whereas co-localization with WT1 predicted dismal prognosis. These results demonstrate that quantitative and structural dimensions of clonality refine the biological and prognostic landscape of NPM1-mutated AML beyond mutational status alone.

Blood
Princess Margaret Cancer Centre (CA), Hospital Universitari i Politècnic La Fe (ES), Research Institute Hospital 12 de Octubre (ES), Hospital Universitario Virgen Macarena (ES), Hospital General Universitario Morales Meseguer (ES), Instituto Maimónides de Investigación Biomédica de Córdoba (ES), Complejo Hospitalario de Navarra (ES), Hospital Universitario de Gran Canaria Doctor Negrín (ES), Marqués de Valdecilla University Hospital (ES), Hospital Universitario 12 De Octubre (ES), Hospital Universitario Quirónsalud Madrid (ES), Hospital San Pedro de Alcántara (ES), Leitat Technological Center (ES), Hospital Universitario Reina Sofía (ES), Hospital General Universitari de Castelló (ES), Hospital General Universitario De Valencia (ES), Hospital Juan Ramón Jiménez (ES), Instituto de Biomedicina de Sevilla (ES), Hospital Universitario Central de Asturias (ES), Clinica Universidad de Navarra (ES), Hospital Clínico Universitario Lozano Blesa (ES), Hospital General Universitario de Alicante Doctor Balmis (ES), Centro de Investigación del Cáncer (ES), Donostiako Unibertsitate Ospitalea (ES), Complexo Hospitalario Universitario A Coruña (ES), Hospital General Universitario de Albacete (ES), Hospital Universitario de Valme (ES), Hospital Universitario Virgen de la Arrixaca (ES), Complejo Hospitalario Torrecárdenas (ES), Hospital Universitario Virgen del Rocío (ES), Hospital Jerez Puerta del Sur (ES), Hospital Clínico Universitario Virgen de la Victoria (ES), INCLIVA Health Research Institute (ES), Hospital Universitario de León (ES), Instituto de Investigación Sanitaria La Fe (ES), Universidad de Sevilla (ES), Universidad de Navarra (ES)
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