Detection of clinically relevant variants in the TP53 gene below 10% allelic frequency: A multicenter study by ERIC, the European Research Initiative on CLL
Authors: Pavlova Sarka , Malcikova Jitka , Radova Lenka , Bonfiglio Silvia , Cowland Jack B. , Brieghel Christian , Andersen Mette K. , Karypidou Maria , Biderman Bella , Doubek Michael , Lazarian Gregory , Rapado Inmaculada , Vynck Matthijs , Porret Naomi A. , Andres Martin , Rosenberg Dina , Sahar Dvora , Martínez‐Laperche Carolina , Buño Ismael , Hindley Andrew , Donaldson David , Sánchez Julio B. , García‐Marco José A. , Serrano‐Alcalá Alicia , Ferrer‐Lores Blanca , Fernández‐Rodriguez Concepción , Bellosillo Beatriz , Stilgenbauer Stephan , Tausch Eugen , Nikdin Hero , Quinn Fiona , Atkinson Emer , van de Corput Lisette , Yildiz Cafer , Bilbao‐Sieyro Cristina , Florido Yanira , Thiede Christian , Schuster Caroline , Stoj Anastazja , Czekalska Sylwia , Chatzidimitriou Anastasia , Laidou Stamatia , Bidet Audrey , Dussiau Charles , Nollet Friedel , Piras Giovanna , Monne Maria , Smirnova Svetlana , Nikitin Eugene , Sloma Ivan , Claudel Alexis , Largeaud Laetitia , Ysebaert Loïc , Valk Peter J. M. , Christian Amy , Walewska Renata , Oscier David , Sebastião Marta , da Silva Maria Gomes , Galieni Piero , Angelini Mario , Rossi Davide , Spina Valeria , Matos Sónia , Martins Vânia , Stokłosa Tomasz , Pepek Monika , Baliakas Panagiotis , Andreu Rafa , Luna Irene , Kahre Tiina , Murumets Ülle , Pikousova Tereza , Kurucova Terezia , Laird Sophie , Ward Daniel , Alcoceba Miguel , Balanzategui Ana , Scarfo Lydia , Gandini Francesca , Zapparoli Ettore , Blanco Adoración , Abrisqueta Pau , Rodríguez‐Vicente Ana E. , Benito Rocío , Bravetti Clotilde , Davi Frédéric , Gameiro Paula , Martinez‐Lopez Joaquin , Tazón‐Vega Bárbara , Baran‐Marszak Fanny , Davis Zadie , Catherwood Mark , Sudarikov Andrey , Rosenquist Richard , Niemann Carsten U. , Stamatopoulos Kostas , Ghia Paolo , Pospisilova Sarka 
Published: January 2025

In chronic lymphocytic leukemia, the reliability of next‐generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low‐VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut‐off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low‐VAF variants on time‐to‐second‐treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low‐VAF TP53 variants had shorter TTST and OS versus wt‐TP53 patients, and the relative risk of second‐line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low‐VAF TP53 variants and wt‐TP53 patients, while patients with high‐VAF TP53 variants had inferior OS compared to wild type‐TP53 cases. Altogether, NGS‐based approaches are technically capable of detecting low‐VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set‐up. Finally, whereas low‐VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment.