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.

AgenDix GmbH Dresden Germany

B Cell Neoplasia Unit and Strategic Research Program on CLL IRCCS Ospedale San Raffaele Milan Italy

Cancer Molecular Diagnostics Department Centre for Laboratory Medicine and Molecular Pathology St James Hospital Dublin Ireland

Center for Omics Sciences IRCCS Ospedale San Raffaele Milan Italy

Central Diagnostic Laboratory University Medical Center Utrecht Utrecht The Netherlands

Centre for Molecular Medicine Central European Institute of Technology Masaryk University Brno Czech Republic

Departement d'Hematologie et Immunologie Biologique AP HP Hopital Henri Mondor Creteil France

Department of Biological Hematology Sorbonne Université AP HP Pitié Salpêtrière Hospital Paris France

Department of Cell Biology Medical School Complutense University of Madrid Madrid Spain

Department of Clinical Genetics and Genomics Karolinska University Hospital Stockholm Sweden

Department of Clinical Genetics Centre of Diagnostic Investigations Copenhagen University Hospital Rigshospitalet Copenhagen Denmark

Department of Hematological Diagnostics and Genetics University Hospital in Krakow Krakow Poland

Department of Hematology and Central Hematology Laboratory Inselspital Bern University Hospital University of Bern Bern Switzerland

Department of Hematology Copenhagen University Hospital Rigshospitalet Copenhagen Denmark

Department of Hematology Erasmus MC Cancer Institute University Medical Center Rotterdam Rotterdam The Netherlands

Department of Hematology Gregorio Marañón General University Hospital Gregorio Marañón Health Research Institute Madrid Spain

Department of Hematology Hospital Universitari Vall d'Hebron Barcelona Spain

Department of Hematology Hospital Universitario 12 de Octubre Instituto de Investigación Sanitaria Hospital 12 de Octubre Complutense University CNIO CIBERONC Madrid Spain

Department of Hematology University Hospital of Salamanca Salamanca Spain

Department of Human Anatomy and Histology Faculty of Medicine University of Salamanca Salamanca Spain

Department of Immunology Genetics and Pathology Uppsala University Uppsala Sweden

Department of Internal Medicine 3 Ulm University Ulm Germany

Department of Internal Medicine Hematology and Oncology and Institute of Medical Genetics and Genomics University Hospital Brno and Medical Faculty Masaryk University Brno Czech Republic

Department of Laboratory Genetics Genetics and Personalized Clinic Tartu University Hospital Tartu Estonia

Department of Laboratory Medicine AZ Sint Jan Hospital Bruges Belgium

Department of Medicine Universitat Autònoma de Barcelona Barcelona Spain

Department of Molecular Medicine and Surgery Karolinska Institutet Stockholm Sweden

Department of Tumor Biology and Genetics Medical University of Warsaw Warsaw Poland

Experimental Hematology Vall d'Hebron Institute of Oncology Barcelona Spain

Genetics and Personalized Medicine Clinic Institute of Clinical Medicine Tartu University Tartu Estonia

Genomed Diagnósticos de Medicina Molecular iMM Instituto de Medicina Molecular Faculdade de Medicina Lisboa Portugal

Genomics Unit Gregorio Marañón General University Hospital Gregorio Marañón Health Research Institute Madrid Spain

Haematology Department Belfast City Hospital Belfast UK

Hematology Department Hospital Clínico Universitario INCLIVA Valencia Spain

Hematology Department Hospital Universitari i Politècnic la Fe Valencia Spain

Hematology laboratory HUPSSD Hôpital Avicenne APHP Bobigny France

Hematology Laboratory Rambam Medical Center Haifa Israel

INSERM U978 Université Sorbonne Paris Nord Bobigny France

Institut Universitaire de Cancérologie de Toulouse Toulouse France

Institute of Applied Biosciences Centre for Research and Technology Hellas Thessaloniki Greece

Institute of Oncology Research and Oncology Institute of Southern Switzerland Bellinzona Switzerland

Laboratoire d'Hématologie Biologique CHU Bordeaux Bordeaux France

Laboratorio di Diagnostica Molecolare Servizio di Genetica Medica EOLAB Ente Ospedaliero Cantonale Bellinzona Switzerland

Laboratório Hemato Oncologia Instituto Português de Oncologia de Lisboa Lisbon Portugal

Laboratorio specialistico UOC ematologia Ospedale San Francesco ASL Nuoro Italy

Medical School Università Vita Salute San Raffaele Milan Italy

Molecular Cytogenetics Unit Hematology Department Hospital Universitario Puerta de Hierro Majadahonda Madrid Spain

Molecular Pathology University Hospitals Dorset Bournemouth UK

National Medical Research Center for Hematology Moscow Russia

Oncohematology Research Group Institute of Biomedical Research of Salamanca and University of Salamanca Salamanca Spain

Outpatient department of Hematology Oncology and Chemotherapy Botkin Hospital and Department of Hematology and Transfusiology Russian Medical Academy of Continuous Professional Education Moscow Russia

Pathology Department Hospital del Mar IMIM Barcelona Spain

Servicio de Hematología Hospital Universitario de Gran Canaria Dr Negrín Departamento de Morfología de La Universidad de Las Palmas de Gran Canaria Gran Canaria Spain

Serviço de Hematologia Instituto Português de Oncologia de Lisboa Lisbon Portugal

Univ Paris Est Creteil INSERM IMRB Creteil France

UOC Hematology Mazzoni Hospital Ascoli Piceno Ascoli Piceno Italy

Wessex Genomics Laboratory Service Salisbury NHS Foundation Trust Salisbury UK

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