TP53 Mutation Analysis in Clinical Practice: Lessons From Chronic Lymphocytic Leukemia

TP53 Mutations are Prognostically Significant in Leukemia and may Accompany Evolution into Higher-Stage Malignancy

In contrary to the majority of solid tumors, the impact of TP53 mutation on patient prognosis in leukemia and lymphoma is unambiguously poor despite being relatively infrequent (5%–15% cases in general) [Robles and Harris, 2010]. The adverse prognosis linked to TP53 defects has been known for a long time [Ahuja et al., 1989; Foti et al., 1990; Slingerland et al., 1991; Sugimoto et al., 1991], and recent detailed analyses of large cohorts together with NGS applications have further deepened our knowledge. Besides CLL, TP53 mutational analysis has been recently suggested as part of prognostication systems also in other hematological disorders—acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) [Bejar et al., 2011; Grossmann et al., 2012].

In AML, TP53 mutations are more common in secondary (post-MPN or post-MDS) disease than in de novo AML, and strongly associate with complex karyotype and higher age. Even if limited to patients with complex karyotype, TP53 mutation is a strong independent predictor of poor survival [Bowen et al., 2009; Milosevic et al., 2012; Rücker et al., 2012]. On the other hand, TP53 mutations are rare in patients with good and intermediate cytogenetics, the subgroups involving the majority of curable patients [Haferlach et al., 2008; Grossmann et al., 2012].

In MDS, TP53 mutations are significantly enriched in patients with chromosome 5 aberrations, both as a sole abnormality (good prognosis group) and as part of complex karyotype (poor prognosis group). Interestingly, the two biologically distinct groups harbor different patterns of TP53 defects: in the group with 5q deletion as a sole abnormality, heterozygous mutations without the loss of the second allele predominantly occur, whereas in patients harboring chromosome 5 defects as part of complex karyotype, both TP53 alleles are usually affected. TP53 mutations in MDS are significantly associated with shortened OS in both aforementioned groups and were disclosed as a strong independent marker for OS compared with commonly used prognostic markers [Kulasekararaj et al., 2013].

TP53 inactivation has also recently been shown as an event accompanying the transformation of myeloproliferative neoplasms (MPN) into AML. Although TP53 mutations have been described only rarely in the chronic phase of MPN, the TP53-mutated clone expands months before transformation [Beer et al., 2010; Harutyunyan et al., 2011].

In chronic myeloid leukemia, TP53 defects are connected with the transformation from chronic to accelerated phase and particularly to blast crisis (BC), where their frequency may reach up to 25%–35% [Calabretta and Perrotti, 2004]. Besides mutations in the TP53 gene, isochromosome 17q resulting in the loss of 17p is commonly observed [Feinstein et al., 1991; Malcikova et al., 2013]. Moreover, in up to 50% of BC-CML patients, TP53 functionality may be impaired by homozygous deletion at the CDKN2A/B genes located on chromosome 9, which codes for a positive TP53 regulator p14^ARF [Sill et al., 1995]. In the era of tyrosine kinase inhibitors, patients rarely undergo blast transformation. The role of TP53 in TKI resistance was also suggested [Wendel et al., 2006], but not reliably proven, as other more relevant mechanisms are likely involved [Malcikova et al., 2013].

In acute lymphoblastic leukemia (ALL), TP53 mutations are more frequent at relapse and are associated with poor therapy response and short OS [Diccianni et al., 1994; Hof et al., 2011; Chiaretti et al., 2013]. It has recently been shown that TP53 mutations are significantly enriched in low-hypodiploid karyotype subgroup, both in adults and children [Holmfeldt et al., 2013]. Importantly, in children, nearly half of the mutations are present in germ-line and consequently ALL in these cases represents a manifestation of Li–Fraumeni syndrome [Felix et al., 1992; Holmfeldt et al., 2013].

In both myeloid and lymphoid malignancies, TP53 dysfunction severely impairs the therapeutic response to chemotherapy [Diccianni et al., 1994; Hof et al., 2011; Rücker et al., 2012; Sellner et al., 2013]. Moreover, using DNA-damaging drugs may impose a strong selection pressure favoring the TP53-mutated subclones [Zhu et al., 1999]. Drugs acting independently of the TP53 pathway represent a treatment of choice if stem cell transplantation is not applicable; however, whether and to what extent TP53-mutated patients may benefit from non-DNA-damaging therapies approved in leukemia is still a matter of intensive research. The prognosis of CLL patients bearing TP53 defects remains poor even if monoclonal antibodies have been used [Sellner et al., 2013]. In low-risk MDS patients treated with lenalidomide, the TP53-mutated subclones were documented to expand during therapy, acquire other abnormalities and this finally lead to AML transformation in some cases [Jädersten et al., 2009; Jädersten et al., 2011].

It is presumed that pathogenic mutations target hematopoietic stem cells (HSCs) and/or early progenitors in neoplastic myeloid disorders and ALL. It seems likely that TP53 mutations contribute to aggressive leukemic stem cell behavior because TP53 protein participates in stem cell homeostasis regulation. Namely, TP53 loss favors symmetric divisions and increases progenitor reprogramming. Moreover, as TP53-mutated HSC clones were described to gain a selective advantage in DNA-damage conditions, TP53-mediated competition may play a role in the early stages of leukemia as well as in its progression [Liu et al., 2009; Bondar and Medzhitov, 2010; Bonizzi et al., 2012]. Although pathologic changes in HSC compartment behavior have also been described in CLL, this mature B-cell neoplasm likely transforms from an as yet unknown subtype of differentiated B-lymphocytes [Kikushige et al., 2011; García-Muñoz et al., 2012; Seifert et al., 2012]. Instead of early hematopoiesis deregulation observed in myeloid neoplasms and acute leukemia, the antigen-driven expansion of B-lymphocytes in the secondary lymphoid tissue microenvironment seems to be a driving force in CLL and lymphoma cancerogenesis. Regardless of the different origin, the impact of TP53 inactivation in CLL is at least as deleterious as in other leukemias.

CLL—TP53 Defects Predispose to Aggressive Disease

CLL represents the most common adult leukemia in western countries, mostly affecting elderly individuals over 50 years of age. The disease is characterized by an extremely heterogeneous clinical course. In contrast to other types of leukemia, the treatment is not initiated upon diagnosis, but upon progression to the symptomatic disease. Some patients may not receive treatment at all and their survival could be comparable to the general population with cause of death often being something other than CLL. On the other hand, there are patients requiring therapy immediately after diagnosis, manifesting an aggressive chemo-refractory disease and overall median survival of only 3–4 years [Hallek, 2013]. Remarkable progress in CLL research that has taken place during the past two decades has led to the identification of molecular prognostic markers allowing early patient stratification according to the expected outcome. Among many prognostic markers published to date, somatic hypermutation presence within the variable region of immunoglobulin heavy-chain gene and TP53 mutations and/or deletions are considered to be the most important. TP53 defects play a crucial role, having an impact not only on progression free survival and OS but also on the response to therapy. Although TP53 abnormalities are relatively infrequent at diagnosis (5%–10%), they may reach up to 40%–50% in the refractory disease. Patients carrying this abnormality are considered to be the most challenging subgroup with respect to therapy intervention and represent an important object of interest in current pharmaceutical research in CLL [Badoux et al., 2011; Sellner et al., 2013].

TP53 Defects in CLL—Both Deletions and Mutations Matter

TP53 gene mutations in CLL were first identified at the beginning of the 90s and already at that time were described in association with advanced disease stage, chemo-refractoriness, and poor clinical outcome [Gaidano et al., 1991; Fenaux et al., 1992; El Rouby et al., 1993]. However, the principal study that introduced TP53 defects into everyday clinical practice was by Dohner et al. (2000). This study established a so-called hierarchical CLL patient classification according to the presence of cytogenetic abnormalities [Dohner et al., 2000]. Interphase in situ hybridization (I-FISH) was used to characterize the recurrent CLL-related chromosomal abnormalities and this analysis clearly showed that patients with deletions of 17p13 containing TP53 locus (del(17p)) have significantly shorter median OS (32 months) compared with patients with normal karyotype (111 months), but also those with other recurrent chromosomal aberrations (deletion 11q–79 months, trisomy 12–114 months, deletion 13q–133 months). The adverse prognostic impact of del(17p) was confirmed later in other studies [Krober et al., 2002; Oscier et al., 2002] and also in clinical trials [Grever et al., 2007; Stilgenbauer et al., 2008; Oscier et al., 2010]. Despite several cases with del(17p) and indolent disease course were described [Best et al., 2009; Tam et al., 2009], this abnormality is considered to be the most adverse prognostic factor and its presence is routinely tested in the clinic. Introducing del(17p) examination into general clinical practice moved TP53 mutation analysis from the scope of interest for a certain period and there were several reasons for this: (1) it was assumed that the correlation between deletion and mutation of the second allele is high in cancer cells [Baker et al., 1990]; (2) the assessment of del(17p) using standardized I-FISH is relatively easy, sensitive, and quantitative, moreover, other prognostically relevant recurrent chromosomal abnormalities are detected in parallel; (3) examining TP53 gene mutations is more complicated and no standardized approach for CLL patients has been established. Attention was attracted back to TP53 mutations in CLL after several studies had shown that quite a large proportion of patients carry TP53 mutation in the absence of del(17p) [Zenz et al., 2008b; Dicker et al., 2009; Malcikova et al., 2009; Rossi et al., 2009]. This resulted in a unique situation when information on both TP53 gene alleles was available for large series of CLL patients, which allowed the allele status and its clinical impact to be studied. The frequency of mutations lacking del(17p) varies among different studies depending on patient cohort and the methodology used, but in general it represents ∼30% of all TP53 defects while sole 17p deletions with the absence of TP53 mutation are less frequent (∼10% of all TP53 defects). The high proportion of TP53 mutation in the absence of del(17p) detected using I-FISH may be in part attributed to the presence of two TP53 mutations on individual alleles in CLL cells. In some patients, TP53 locus copy number neutral loss of heterozygosity (uniparental disomy) was described, which results in duplication of the mutant allele [Saddler et al., 2008]. Nevertheless, monoallelic TP53 defects unequivocally exist in CLL, and sole mutations are more frequent than sole deletions of the whole TP53 locus. Whether the monoallelic TP53 defect impact the prognosis of CLL patients to the same extent as the biallelic defect was the question that attracted many groups. Indeed, it was confirmed that both sole mutations and sole deletions significantly reduced time to first treatment, progression free survival, and also OS [Zenz et al., 2008b; Dicker et al., 2009; Malcikova et al., 2009; Rossi et al., 2009]. The independent negative prognostic impact of TP53 mutations was also subsequently confirmed in prospective clinical trials [Zenz et al., 2009; Zenz et al., 2010a; Gonzalez et al., 2011]. This was in fact an in vivo confirmation of a phenomena described so far only in in vitro studies and mouse models—haploinsufficiency, dominant negative effect, and gain-of-function of specific TP53 mutants [Donehower et al., 1992; Blagosklonny, 2000]. The presence of any TP53 defect holds its prognostic significance even in the era of novel gene mutations in CLL revealed by NGS (e.g., in genes BIRC3, NOTCH1, and SF3B1), and based on the most recently published integrated mutational and cytogenetic model for classifying newly diagnosed CLL patients according to risk of death, patients with TP53 abnormalities are assessed as a “high-risk” subgroup with the shortest OS [Rossi et al., 2013].

In addition to TP53 defects, deletions and mutations in ATM kinase gene, which is an upstream TP53 activator, frequently occur in CLL and have been shown to negatively impact progression free and OS [Pettitt et al., 2001; Skowronska et al., 2012; Stankovic and Skowronska, 2013]. Although ATM deletion (del(11q)) is routinely analyzed by I-FISH, mutations in the ATM gene are rarely examined because of an extreme gene length [Navrkalova et al., 2013].

Spectrum of TP53 Mutations in CLL and Genotype–Phenotype Correlation

The spectrum of TP53 mutations identified in CLL patients is similar to other cancers, but some disease-specific characteristics were identified [Newcomb et al., 1995; Zenz et al., 2010b]. A large international collaborative study analyzing 268 mutations from four independent cohorts [Zenz et al, 2010b] showed a lower proportion of transitions at CpG sites compared with other cancers (e.g., colon cancer), which was not attributed to the decreased number of all transitions. More interestingly, the prevalent G>A versus C>T transitions at CpG sites were observed (1.9:1). Similar data may also be observed for CLL in the IARC TP53 Database (R16, November 2012) [Petitjean et al., 2007], where the G>A versus C>T ratio is even higher 2.45:1, whereas other cancers showed a closer ratio (e.g., breast and colon cancer 1.4:1). It was suggested that this G>A prevalence might reflect the preferential selection of mutations arising from cytosine deamination at the transcribed strand, which has been observed in nondividing cells [Rodin and Rodin, 1998].

The majority of mutations are localized within the DNA-binding domain, and about ∼75% mutations are missense mutations. Truncating mutations were observed significantly more frequently outside the DNA-binding domain (52% vs. 18%), and also in patients with del(17p) when compared with patients with sole mutations (23% vs. 13%) [Zenz et al., 2010b]. A similar disproportion was also noted in patients with Li–Fraumeni syndrome. In patients carrying germline truncating mutations, a selection pressure exists favoring the second allele loss during tumor formation, whereas in cases with missense mutations, the second allele is lost less frequently. This is explained by the dominant negative and gain-of-function phenotype of missense mutations [Varley, 2003]. Moreover, the gain-of-function phenomenon was also clinically proved by our study documenting an extremely adverse prognosis for patients with mutations localized within the DNA-binding motifs (DBMs), the parts of DNA binding domain that are directly involved in contact with DNA (loops L2, L3, and loop-sheet-helix motif). Patients carrying missense mutations in DBMs had clearly shorter time to first treatment and OS compared with both remaining missense mutations and nonmissense alterations (Fig. 1). This was confirmed in a subgroup limited to patients with the accompanying del(17p), which is crucial, as mutated TP53 gain-of-function should be rigorously studied in the absence of wild-type TP53 [Trbusek et al., 2011].

TP53 Defects Impair Therapeutic Response of CLL Patients

As mentioned previously, the standard of care in CLL patients is the “watch and wait” approach, which means that only symptomatic patients are treated, and about half of CLL patients never require treatment. Unfortunately, all patients who require therapy intervention are expected to relapse as CLL remains, despite progressive research, an incurable disease. The current standard treatment is based on alkylation agents and nucleoside analogues combined with immunotherapy, which induces long-term remissions in the majority of patients and prolongs OS. However, this does not hold true for patients with TP53 defects as these are often resistant to standard chemoimmunotherapy and their OS is still very short when compared with other CLL patients (∼3 years) [Hallek et al., 2010]. It is quite obvious that patients with TP53 defects should be treated avoiding DNA-damaging chemotherapy agents, but only limited options exist for these patients in general clinical practice. Despite using agents acting independently on the TP53 pathway (e.g., specific monoclonal antibodies or corticosteroids), the response duration in these patients is still very short [Thornton et al., 2003; Hillmen et al., 2007]. This is most likely caused by the fact that none of the therapeutics currently used are capable of eradicating the disease completely and small subclones of CLL cells hidden in a microenvironment would expand more rapidly when having a TP53 defect. For these reasons, patients with defective TP53 may be considered for allogeneic stem cell transplantation, which is the only curative approach in CLL. However, transplantation is inappropriate for the majority of patients because of their higher age, comorbidities, and also the risk of transplantation-related mortality [Dreger et al., 2010]. All in all, there is currently no optimal treatment available for CLL patients with 17p deletions and/or TP53 mutations. A number of novel biological compounds acting via diverse TP53-independent mechanisms of action are in various phases of clinical or preclinical testing (e.g., B-cell receptor signaling inhibitors) and patients carrying TP53 abnormality should be scheduled to participate in such trials whenever possible [Badoux et al., 2011; Sellner et al., 2013].

Examining TP53 Defects in CLL Clinical Practice

Among a number of prognostic markers used in CLL, TP53 gene defects are currently the only genetic factors that can predict treatment response and influence the choice of therapy [Sellner et al., 2012]. Therefore, the recommendation to examine for del(17p) at least in clinical trials was also included in the updated guidelines for CLL diagnosis and treatment published in the year 2008 [Hallek et al., 2008] and I-FISH is recommended as the standardized methodology. The cutoff for a positive result varies in different laboratories, but generally I-FISH is able to detect more than 5% of del(17p) positive cells. The clinically relevant threshold has also been discussed and it was shown that the presence of >20%–25% of cells with del(17p) represents the most efficient cutoff predicting the patients’ outcome [Catovsky et al., 2007; Tam et al., 2009]. For research use, information on additional chromosomal changes may be obtained using SNP-arrays allowing the detection of copy number neutral loss of heterozygozity and complex karyotype, which was described to be associated with TP53 defects [Ouillette et al., 2013].

As many studies proved the independent impact of TP53 mutations in the absence of del(17p) (see above), recommendations on mutation analysis in CLL were recently published [Pospisilova et al., 2012] and thus, examining both mutations and deletions is currently recommended in clinical practice at least before any treatment initiation in CLL patients. For TP53 mutation detection, there are several methodological possibilities, with each of them having advantages and shortcomings. Although Sanger sequencing is a relatively simple methodology currently widely available in the majority of laboratories, it may not be sufficient to detect TP53-mutated subclones below ∼15%–20%, especially in cases without the deletion of the second allele. Some laboratories employ prescreening methods, for example, DHPLC, which requires specific instrumentation, or functional analysis FASAY (Functional Analysis in Separated Alleles in Yeast) [Flaman et al., 1995; Smardova et al., 2002]. FASAY is also a relatively cheap and simple screening methodology providing a sufficient detection limit, however it may omit some mutations leading to nonsense-mediated RNA decay. Technological progress within the last few years has led to the introduction of modern genomic technologies, namely DNA arrays and NGS, both in the wider scientific community and also in the clinic. Particularly NGS represents the most likely tool to be utilized in the near future (see below).

Clonal Evolution of TP53 Defects Occurs Mainly under Therapy Pressure

At diagnosis or before the first therapy, only 5%–15% of CLL patients, depending on the method used and patients’ cohort, are reported to carry TP53 defect [Rossi et al., 2009; Zenz et al., 2010a; Gonzalez et al., 2011; Zainuddin et al., 2011]. However, the frequency of TP53 defects increases substantially during the disease course and reaches up to 40% in patients that are refractory to purine analogues. Moreover, each second patient with CLL transformation to more aggressive prolymphocytic leukemia [Lens et al., 1997] or diffuse large B cell lymphoma (Richter transformation) harbor TP53 disruption [Rossi et al., 2011]. The del(17p) and/or TP53 mutation acquisition was documented in individual cases by several studies analyzing sequential patients’ samples. The expansion of CLL clones carrying these defects was strongly associated with foregoing therapy [Stilgenbauer et al., 2007; Zenz et al., 2008a; Zenz et al., 2008b; Malcikova et al., 2009; Rossi et al., 2009; Ouillette et al., 2013], whereas the acquisition of a novel TP53 defect in untreated patients was observed only rarely [Ouillette et al., 2013]. It is therefore widely accepted that treatment plays a key role in TP53 defect selection, or as originally suggested, may possibly contribute to the acquisition of these defects through direct DNA mutagenesis (Fig. 2) [Newcomb et al., 1995; Sturm et al., 2003]. Indeed in some cancers, the role of environmental carcinogens was documented and the TP53 mutations in these cancers showed a specific profile [Hernandez-Boussard and Hainaut, 1998; Hussain et al., 2007]. However, the comparison of TP53 mutation profiles in CLL patients with and without previous therapy showed no difference [Zenz et al., 2010b] suggesting that the TP53 mutations detectable after therapy are selected rather than directly induced by therapeutic agents (Fig. 2). To reliably confirm this “selection” hypothesis, the thorough analysis of pretreatment samples is necessary. The first study retrospectively searching for the later selected TP53 mutations confirmed their presence in two out of three pretreatment samples using subcloning in bacteria [Zenz et al., 2008b]. Notwithstanding, the opportunity to study the clonal evolution of mutations comes with the era of NGS as this technology allows high-throughput ultradeep analysis with an as yet unattainable sensitivity below 1% of mutated alleles in a sample.

TP53 Mutational Analysis using NGS

NGS, also called massively parallel sequencing or high-throughput sequencing, is generally defined as a technology that employs parallel sequencing processes producing tens of thousands to billions of sequence reads simultaneously. Unlike Sanger sequencing, DNA molecules in the mixture are sequenced separately. Current NGS applications in cancer research and diagnostics enable analyses ranging from several genes (“targeted resequencing”) to whole exomes or even genomes [Mardis, 2008; Schuster, 2008; Metzker, 2010]. The platforms currently used require clonally amplified DNA for signal detection; however, development is moving toward a single molecule template, thus eliminating the risk of artificial “mutations” created by PCR amplification [Loman et al., 2012]. Each platform is connected with particular advantages and shortcomings that should be taken into consideration, namely different read lengths, the capability to detect small insertions and deletions in homopolymer regions, sequencing capacity, and read quality.

Unique “barcode” sequences (indexes) can be added to each sample so they are distinguishable during data analysis, bringing the possibility to analyze many samples in one run. When a larger number of patient samples or multiple sequences (genes, exons) are analyzed, NGS represents a cheaper and less time-consuming option than other methods routinely used in diagnostics. Besides the possibility to optimize the sample preparation process, several currently available commercial kits enable amplification of TP53 and other disease-related genes in separate or multiplex PCR reactions. TP53 is also included in multiple commercially available cancer panels, which can be flexibly used with diverse sensitivity. According to expected outputs, the sequencer capacity can be used to analyze either a high number of samples in parallel, or a few samples with very high sensitivity (“deep sequencing”). Deep or ultradeep sequencing enables the detection of very low-abundant sequence variants, which permits previously impossible applications in oncology, noninvasive prenatal diagnostics, and other fields [Kohlmann et al., 2011; Kohlmann et al., 2013]. Of note, the term “deep sequencing” has not been exactly defined and is used for a wide sensitivity range determined by an extremely variable number of sequencing reads ranging from hundreds to tens of thousands.

The main challenge of deep sequencing lies in result interpretation, namely distinguishing true mutations from random errors [Meacham et al., 2011; Flaherty et al., 2012]. Thus, the sensitivity of small clone detection depends on: (1) a sufficient number of input DNA(RNA) molecules in the sample (cell equivalent), (2) the number of molecules being read for the sample (number of “reads”), generally described as “coverage,” (3) the background level of error rate introduced during the sample preparation (polymerase errors), sequencing process, and data analysis (alignment errors). The use of proofreading enzymes significantly improves detection limits ([Vandenbroucke et al., 2011] and our unpublished data). So far no recommendations regarding the statistical analysis of deep sequencing data and, in particular, sensitivity definition exist. Several statistical approaches with variable postulates regarding error rates for each nucleotide position have been applied [Flaherty et al., 2012; Wilm et al., 2012; Grossmann et al., 2013]. Generally, searching for a specific mutation in paired-sample analysis, for example, monitoring minimal residual disease or retrospective tracking of minor mutated subclones, is supposed to reach a higher sensitivity. When changes other than point mutations are backtracked, it is presumed that exactly the same mutation is less likely to arise as an amplification error, which permits to improve detection limit [Kohlmann et al., 2013]. Unfortunately this is not the case for TP53 where missense mutations predominate.

In oncology, deep sequencing TP53 analysis allows the detection of small mutTP53-bearing subclones resistant to therapy, which may give rise to tumor cell population expanding at relapse. Thus, the detection of the presence of minor TP53-mutated subclone may be potentially desirable. However, apart from the technical aspects described above, the interpretation of minor TP53-mutated clone presence and its implications for patient care are currently widely unknown and these analyses are in progress.

In CLL, the sensitivity of the methods routinely used to detect TP53 defects ranges from 5% to 20%, but the pathogenic impact of TP53 defects present in less than 20% of tumor cells is still a matter of debate [Catovsky et al., 2007; Tam et al., 2009]. The situation is likely to be much more complex in cases of clones not detectable by standard methods. Although published data are limited, it is likely that expansion, persistence, and disappearance of minor clones may occur (Figs. 2 and 3). In a proportion of patients, the minor TP53-defective clone may be relatively stable during the disease course, at least in untreated patients [Jethwa et al., 2013], and in some cases, even in spite of repeated therapies (our unpublished observation). The expansion of TP53-mutated subclones in relapse has been repeatedly described retrospectively [Zenz et al., 2008a; Zenz et al., 2008b; Trbusek et al., 2012; Jethwa et al., 2013]. In our cohort of more than 200 patients examined with FASAY coupled to Sanger sequencing repeatedly during the disease course, we observed that approximately each fifth treated patient acquired a novel TP53 mutation. In nine patients, the mutations were backtracked using NGS, and in all cases, their presence was already proven in a minor clone (0.3%–13% of mutated alleles) before first therapy. Our analysis also showed the occurrence of multiple TP53-mutated clones in several patients (an illustrative case is shown in (Fig. 3) [Trbusek et al., 2012]. Similar results confirming the presence of convergent mutations were reported very recently [Jethwa et al., 2013]. Thus, modern technologies provide the opportunity to identify patients at risk of TP53 defective clone expansion; however, how frequently these minor clones undergo expansion is currently unknown and this issue should be intensively studied in prospective studies.