REVIEW ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia S Pospisilova 1,14 , D Gonzalez 2,14 , J Malcikova 1 , M Trbusek 1 , D Rossi 3 , AP Kater 4 , F Cymbalista 5 , B Eichhorst 6 , M Hallek 6 , H Do ¨ hner 7 , P Hillmen 8 , M van Oers 4 , J Gribben 9 , P Ghia 10 , E Montserrat 11 , S Stilgenbauer 7 and T Zenz 12,13 on behalf of the European Research Initiative on CLL (ERIC) Recent evidence suggests that -- in addition to 17p deletion -- TP53 mutation is an independent prognostic factor in chronic lymphocytic leukemia (CLL). Data from retrospective analyses and prospective clinical trials show that B5% of untreated CLL patients with treatment indication have a TP53 mutation in the absence of 17p deletion. These patients have a poor response and reduced progression-free survival and overall survival with standard treatment approaches. These data suggest that TP53 mutation testing warrants integration into current diagnostic work up of patients with CLL. There are a number of assays to detect TP53 mutations, which have respective advantages and shortcomings. Direct Sanger sequencing of exons 4--9 can be recommended as a suitable test to identify TP53 mutations for centers with limited experience with alternative screening methods. Recommendations are provided on standard operating procedures, quality control, reporting and interpretation. Patients with treatment indications should be investigated for TP53 mutations in addition to the work-up recommended by the International workshop on CLL guidelines. Patients with TP53 mutation may be considered for allogeneic stem cell transplantation in first remission. Alemtuzumab-based regimens can yield a substantial proportion of complete responses, although of short duration. Ideally, patients should be treated within clinical trials exploring new therapeutic agents. Leukemia advance online publication, 6 March 2012; doi:10.1038/leu.2012.25 Keywords: TP53; CLL; mutation INTRODUCTION The tumor suppressor p53 has a crucial role in cellular response to stress or DNA damage by induction of cell-cycle arrest, apoptosis or senescence. 1 Altered p53 function because of 17p deletion and/or TP53 gene mutation is associated with poor prognosis in chronic lymphocytic leukemia (CLL) patients. 2-7 Aberrations of TP53 gene occur on average in 10--15% of untreated CLL patients, but the incidence rises to 40--50% with fludarabine-refractory CLL. 8,9 Over 80% of cases with 17p deletion also carry TP53 mutations in the remaining allele. 2,4 - 6 Recently it has been shown that TP53 mutations in the absence of 17p deletion occur in a significant proportion of CLL patients (B5% in first line treatment situation) and are associated with significantly worse outcome. 3,5 Table 1 shows the impact of TP53 mutations on outcome in recent prospective clinical trials. The aim of this report is to provide recommendations on TP53 mutation analysis in patients with CLL. Recommendations concerning several methodologies suitable for TP53 analysis will be provided, including comments on their respective advantages, shortcomings and clinical utility. We will also discuss potential clinical consequences derived from the positive/negative results, although a full clinical review is beyond the scope of this article. TP53 MUTATION IN CLL Mutations represent the most frequent form of p53 inactivation in CLL and are frequently accompanied by the loss of the second allele (through 17p deletion, or more rarely copy number neutral loss of heterozygosity). 2,10 - 12 In total, 95% of mutations are localized within the central DNA-binding domain, impairing DNA binding and target gene transactivation. 13 Approximately 75% of all mutations represent missense mutations leading to amino-acid change. A significant proportion of all mutations are localized in classic hot-spot codons. These mutations either directly disrupt the p53-DNA interaction (‘DNA-contact mutants’---e.g., residues 248 and 273) or cause conformational changes (‘conformational mutants’---e.g., residues 175, 245, 249and 282). 14 The vast majority of mutations lead to severely impaired p53 function. 15 However, not all missense mutations convey a complete loss of function and Received 26 January 2012; accepted 27 January 2012; accepted article preview online 2 February 2012 1 Central European Institute of Technology, Masaryk University and Department of Hematology and Oncology, University Hospital Brno, Brno, Czech Republic; 2 Division of Molecular Pathology, The Institute of Cancer Research, London, UK; 3 Department of Translational Medicine, Division of Hematology, Amedeo Avogadro University of Eastern Piedmont, Novara, Italy; 4 Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands; 5 Ho ˆ pital Avicenne, Universite ´ Paris XIII, Laboratoire d’he ´ matologie, Bobigny, France; 6 Department of Internal Medicine I, Center for Integrated Oncology Ko ¨ ln-Bonn, University of Cologne, Cologne, Germany; 7 Department of Internal Medicine III, University of Ulm, Ulm, Germany; 8 Leeds Teaching Hospitals NHS Trust, St James’s University Hospital, Leeds, UK; 9 Centre for Haemato-Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK; 10 Laboratory of B-cell Neoplasia, Division of Molecular Oncology, San Raffaele Scientific Institute, Milan, Italy; 11 Department of Hematology, Institute of Hematology and Oncology, Hospital Clinic, Barcelona, IDIBAP, University of Barcelona, Barcelona, Spain; 12 Department of Translational Oncology, National Center for Tumor Diseases (NCT), German Cancer Research Center (DKFZ), Heidelberg, Germany and 13 Department of Internal Medicine V, University Hospital Heidelberg, Heidelberg, Germany. Correspondence: Dr T Zenz, Abteilung fu ¨ r Translationale Onkologie, Nationales Centrum fu ¨ r Tumorerkrankungen (NCT) Heidelberg und Deutsches, Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 460, Heidelberg 69120, Germany. E-mail: thorsten.zenz@nct-heidelberg.de 14 These authors contributed equally to this work. Leukemia (2012) 1 - 4 & 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12 www.nature.com/leu