Laboratories Can Reliably Detect Clinically Relevant Variants in the TP53 Gene below 10 % Allelic Frequency: A Multicenter Study of ERIC, the European Research Initiative on CLL

• Published in: BLOOD 2023-11, Vol.142 (Supplement 1), p.200-200
• Main Authors: Pavlova, Sarka, Malcikova, Jitka, Radova, Lenka, Bonfiglio, Silvia, Cowland, Jack B., Brieghel, Christian, Andersen, Mette Klarskov, Karypidou, Maria, Biderman, Bella V, Doubek, Michael, Lazarian, Gregory, Rapado, Inmaculada, Vynck, Matthijs, Porret, Naomi, Andres, Martin, Rosenberg, Dina, Sahar, Dvora, Martinez-Laperche, Carolina, Borde, Ismael Buño, Hindley, Andrew, Sánchez, Julio Bravo, García-Marco, José, Serrano, 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, Cristina, Florido, Yanira, Thiede, Christian, Schuster, Caroline, Stoj, Anastazja, Czekalska, Sylwia, Chatzidimitriou, Anastasia, Laidou, Stamatia, Bidet, Audrey, Dussiau, Charles, Nollet, Friedel, Piras, Giovanna, Borosova, Tereza, Kurucova, Terezia, Monne, Maria, Smirnova, Svetlana, Nikitin, Evgeny, Sloma, Ivan, Delfau, Marie-Helene, Largeaud, Laetitia, Ysebaert, Loic, Valk, Peter J. M., Christian, Amy, Walewska, Renata, Sebastião, Marta, da Silva, Maria Gomes, Galieni, Piero, Angelini, Mario, Rossi, Davide, Spina, Valeria, Matos, Sónia, Martins, Vânia, Donaldson, David, Stoklosa, Tomasz, Pepek, Monika, Baliakas, Panagiotis, Andreu, Rafa, Luna, Irene, Kahre, Tiina, Murumets, Ülle, Laird, Sophie, Ward, Daniel, Alcoceba, Miguel, Balanzategui, Ana, Scarfo, Lydia, Gandini, Francesca, Zapparoli, Ettore, Blanco, Adoracion, Costa, Pau Abrisqueta, Rodriguez, Ana E., Benito Sanchez, Maria Rocio, Davi, Frédéric, Bravetti, Clothilde, Gameiro, Paula, Martinez-Lopez, Joaquin, Tazon, Barbara, Baran-Marszak, Fanny, Davies, Zadie, Caterwood, Mark, Sudarikov, Andrey B, Rosenquist, Richard, Niemann, Carsten Utoft, Stamatopoulos, Kostas, Ghia, Paolo, Pospisilova, Sarka
• Format: Article
• Language: English
• Subjects: Medicin och hälsovetenskap
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2023-173235

The presence of mutations in the TP53 gene is a powerful prognostic and predictive marker in chronic lymphocytic leukemia (CLL). Widespread use of NGS has enabled the detection of variants ≤10 % variant allelic frequency (low-VAF variants); however, the overall reliability and reproducibility of NGS techniques to identify such variants have been questioned repeatedly. Individual studies using sensitive, custom NGS-based assays have mostly demonstrated the shortened overall survival (OS) and event-free survival in patients with low-VAF TP53 variants treated with chemoimmunotherapy (CIT) regimens with median survival ranging between that of TP53 variants >10 % VAF (high-VAF) and wild-type TP53 (wt- TP53). Within an ERIC multicenter study, we tested the ability of NGS methods used in diagnostic and research laboratories to detect low-VAF TP53 variants and analyzed the impact of the identified low-VAF variants on patients' survival. In the first phase of the study (Fig. 1), seven sample mixes containing 23 pathogenic TP53 variants (range, 0.7-6.3% VAF) were analyzed in 41 ERIC centers using 44 NGS-based assays. All variants were validated with droplet digital PCR (ddPCR); obtained values were used as a reference for the assessment of each NGS method's performance. NGS results were categorized as true positive (TP), false positive (FP; not present in original samples and reported by one center each), and not reported/false negative (FN). In total, laboratories reported 77.8% of all variants (784 out of 1008), reaching a sensitivity [TP/ (TP + FN)] of 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While the VAFs of individual variants reported by laboratories varied, median values strongly correlated with ddPCR (R 2=0.9841). Thirty-eight FP variants were reported by 10 laboratories, mainly 1 and ≤2%, 1 FP > 2%). Individual feedback was provided to improve the methods' performance and to help set an appropriate detection limit. In the second phase of the study, 12 centers provided results of TP53 NGS-based analysis of 1092 CLL clinical samples taken before first-line treatment (median time from sample to treatment 40 days). The impact of low-VAF variants (1-10% VAF; N=59) on time to second treatment (TTST; event: second treatment, death) and OS calculated from 1 st treatment initiation was compared to that of high-VAF variants (N=123) and wt- TP53 using logrank test with Benjamini-Hochberg correction