In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.

] Center of Molecular Medicine Central European Institute of Technology Masaryk University Brno Czech Republic [2] Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

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

Department of Internal Medicine Hematology and Oncology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Department of Pathology University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Stilgenbauer S, Zenz T. Understanding and managing ultra high-risk chronic lymphocytic leukemia. Hematol Am Soc Hematol Educ Program. 2010;2010:481–488. PubMed

Zenz T, Eichhorst B, Busch R, Denzel T, Häbe S, Winkler D, et al. TP53 mutation and survival in chronic lymphocytic leukemia. J Clin Oncol. 2010;28:4473–4479. PubMed

Gonzalez D, Martinez P, Wade R, Hockley S, Oscier D, Matutes E, et al. Mutational status of the TP53 gene as a predictor of response and survival in patients with chronic lymphocytic leukemia: results from the LRF CLL4 trial. J Clin Oncol. 2011;29:2223–2229. PubMed

Byrd JC, Gribben JG, Peterson BL, Grever MR, Lozanski G, Lucas DM, et al. Select high-risk genetic features predict earlier progression following chemoimmunotherapy with fludarabine and rituximab in chronic lymphocytic leukemia: justification for risk-adapted therapy. J Clin Oncol. 2006;24:437–443. PubMed

Badoux XC, Keating MJ, Wang X, O'Brien SM, Ferrajoli A, Faderl S, et al. Fludarabine, cyclophosphamide, and rituximab chemoimmunotherapy is highly effective treatment for relapsed patients with CLL. Blood. 2011;117:3016–3024. PubMed PMC

Stilgenbauer S, Zenz T, Winkler D, Bühler A, Schlenk R, Groner S, et al. Subcutaneous alemtuzumab in fludarabine-refractory chronic lymphocytic leukemia: clinical results and prognostic marker analyses from the CLL2H study of the German Chronic Lymphocytic Leukemia Study Group. J Clin Oncol. 2009;27:3994–4001. PubMed

Pettitt AR, Sherrington PD, Stewart G, Cawley JC, Taylor AM, Stankovic T. p53 dysfunction in B-cell chronic lymphocytic leukemia: inactivation of ATM as an alternative to TP53 mutation. Blood. 2001;98:814–822. PubMed

Ouillette P, Fossum S, Parkin B, Ding L, Bockenstedt P, Al-Zoubi A, et al. Aggressive chronic lymphocytic leukemia with elevated genomic complexity is associated with multiple gene defects in the response to DNA double-strand breaks. Clin Cancer Res. 2010;16:835–847. PubMed PMC

Landau DA, Carter SL, Stojanov P, McKenna A, Stevenson K, Lawrence MS, et al. Evolution and impact of subclonal mutations in chronic lymphocytic leukemia. Cell. 2013;152:714–726. PubMed PMC

Rossi D, Cerri M, Deambrogi C, Sozzi E, Cresta S, Rasi S, et al. The prognostic value of TP53 mutations in chronic lymphocytic leukemia is independent of Del17p13: implications for overall survival and chemorefractoriness. Clin Cancer Res. 2009;15:995–1004. PubMed

Zainuddin N, Murray F, Kanduri M, Gunnarsson R, Smedby KE, Enblad G, et al. TP53 mutations are infrequent in newly diagnosed chronic lymphocytic leukemia. Leuk Res. 2011;35:272–274. PubMed

Zenz T, Häbe S, Denzel T, Mohr J, Winkler D, Bühler A, et al. Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial. Blood. 2009;114:2589–2597. PubMed

Shanafelt TD, Witzig TE, Fink SR, Jenkins RB, Paternoster SF, Smoley SA, et al. Prospective evaluation of clonal evolution during long-term follow-up of patients with untreated early-stage chronic lymphocytic leukemia. J Clin Oncol. 2006;24:4634–4641. PubMed

Stilgenbauer S, Sander S, Bullinger L, Benner A, Leupolt E, Winkler D, et al. Clonal evolution in chronic lymphocytic leukemia: acquisition of high-risk genomic aberrations associated with unmutated VH, resistance to therapy, and short survival. Haematologica. 2007;92:1242–1245. PubMed

Zenz T, Krober A, Scherer K, Habe S, Buhler A, Benner A, et al. Monoallelic TP53 inactivation is associated with poor prognosis in chronic lymphocytic leukemia: results from a detailed genetic characterization with long-term follow-up. Blood. 2008;112:3322–3329. PubMed

Malcikova J, Smardova J, Rocnova L, Tichy B, Kuglik P, Vranova V, et al. Monoallelic and biallelic inactivation of TP53 gene in chronic lymphocytic leukemia: selection, impact on survival, and response to DNA damage. Blood. 2009;114:5307–5314. PubMed

Ouillette P, Saiya-Cork K, Seymour E, Li C, Shedden K, Malek SN. Clonal evolution, genomic drivers, and effects of therapy in chronic lymphocytic leukemia. Clin Cancer Res. 2013;19:2893–2904. PubMed PMC

Rossi D, Khiabanian H, Spina V, Ciardullo C, Bruscaggin A, Famà R, et al. Clinical impact of small TP53 mutated subclones in chronic lymphocytic leukemia. Blood. 2014;123:2139–2147. PubMed PMC

Pospisilova S, Gonzalez D, Malcikova J, Trbusek M, Rossi D, Kater AP, et al. ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia. Leukemia. 2012;26:1458–1461. PubMed

Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, et al. Genomic aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000;343:1910–1916. PubMed

R Core Team A Language and Environment for Statistical Computing R Foundation for Statistical Computing: Vienna, Austria; . http://www.R-project.org/2013

Gerstung M, Papaemmanuil E, Campbell PJ. Subclonal variant calling with multiple samples and prior knowledge. Bioinformatics. 2014;30:1198–1204. PubMed PMC

Gerstung M, Beisel C, Rechsteiner M, Wild P, Schraml P, Moch H, et al. Reliable detection of subclonal single-nucleotide variants in tumour cell populations. Nat Commun. 2012;3:811. PubMed

Zenz T, Vollmer D, Trbusek M, Smardova J, Benner A, Soussi T, et al. TP53 mutation profile in chronic lymphocytic leukemia: evidence for a disease specific profile from a comprehensive analysis of 268 mutations. Leukemia. 2010;24:2072–2079. PubMed

Sturm I, Bosanquet AG, Hermann S, Guner D, Dorken B, Daniel PT. Mutation of p53 and consecutive selective drug resistance in B-CLL occurs as a consequence of prior DNA-damaging chemotherapy. Cell Death Differ. 2003;10:477–484. PubMed

Catovsky D, Richards S, Matutes E, Oscier D, Dyer MJ, Bezares RF, et al. Assessment of fludarabine plus cyclophosphamide for patients with chronic lymphocytic leukaemia (the LRF CLL4 Trial): a randomised controlled trial. Lancet. 2007;370:230–239. PubMed

Tam C, Shanafelt T, Wierda W, Abruzzo L, Van Dyke D, O'Brien S, et al. De novo deletion 17p13.1 chronic lymphocytic leukemia shows significant clinical heterogeneity: the M. D. Anderson and Mayo Clinic experience. Blood. 2009;114:957–964. PubMed PMC

Rossi D, Rasi S, Spina V, Bruscaggin A, Monti S, Ciardullo C, et al. Integrated mutational and cytogenetic analysis identifies new prognostic subgroups in chronic lymphocytic leukemia. Blood. 2013;121:1403–1412. PubMed PMC

Jethwa A, Hüllein J, Stolz T, Blume C, Sellner L, Jauch A, et al. Targeted resequencing for analysis of clonal composition of recurrent gene mutations in chronic lymphocytic leukaemia. Br J Haematol. 2013;163:496–500. PubMed

Puente XS, Pinyol M, Quesada V, Conde L, Ordóñez GR, Villamor N, et al. Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia. Nature. 2011;475:101–105. PubMed PMC

Zhao Y, Gregory MT, Biertümpfel C, Hua YJ, Hanaoka F, Yang W. Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η. Proc Natl Acad Sci USA. 2013;110:8146–8151. PubMed PMC

Panovská A, Smolej L, Lysák D, Brychtová Y, Šimkovič M, Motyčková M, et al. The outcome of chronic lymphocytic leukemia patients who relapsed after fludarabine, cyclophosphamide, and rituximab. Eur J Haematol. 2013;90:479–485. PubMed

Trbusek M, Smardova J, Malcikova J, Sebejova L, Dobes P, Svitakova M, et al. Missense mutations located in structural p53 DNA-binding motifs are associated with extremely poor survival in chronic lymphocytic leukemia. J Clin Oncol. 2011;29:2703–2708. PubMed

Unveiling the dynamics and molecular landscape of a rare chronic lymphocytic leukemia subpopulation driving refractoriness: insights from single-cell RNA sequencing

Splice variants of CK1α and CK1α-like: Comparative analysis of subcellular localization, kinase activity, and function in the Wnt signaling pathway

Role of ciliopathy protein TMEM107 in eye development: insights from a mouse model and retinal organoid

Distinct p53 phosphorylation patterns in chronic lymphocytic leukemia patients are reflected in the activation of circumjacent pathways upon DNA damage

Dual specificity phosphatase 7 drives the formation of cardiac mesoderm in mouse embryonic stem cells

Evolution of TP53 abnormalities during CLL disease course is associated with telomere length changes

Low-burden TP53 mutations in CLL: clinical impact and clonal evolution within the context of different treatment options

Chromothripsis in Chronic Lymphocytic Leukemia: A Driving Force of Genome Instability

RNF43 inhibits WNT5A-driven signaling and suppresses melanoma invasion and resistance to the targeted therapy

Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study

Prognostic and predictive role of gene mutations in chronic lymphocytic leukemia: results from the pivotal phase III study COMPLEMENT1

Protease associated domain of RNF43 is not necessary for the suppression of Wnt/β-catenin signaling in human cells

Performance of anti-CD19 chimeric antigen receptor T cells in genetically defined classes of chronic lymphocytic leukemia

WNT signaling inducing activity in ascites predicts poor outcome in ovarian cancer

Distinct cellular responses to replication stress leading to apoptosis or senescence

The planar cell polarity protein VANG-1/Vangl negatively regulates Wnt/β-catenin signaling through a Dvl dependent mechanism

TP53 aberrations in chronic lymphocytic leukemia: an overview of the clinical implications of improved diagnostics

ToTem: a tool for variant calling pipeline optimization

ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-update on methodological approaches and results interpretation

Low-burden TP53 mutations in chronic phase of myeloproliferative neoplasms: association with age, hydroxyurea administration, disease type and JAK2 mutational status