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 ∼5% 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.
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, 3, 4, 5, 6, 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, 5, 6 Recently it has been shown that TP53 mutations in the absence of 17p deletion occur in a significant proportion of CLL patients (∼5% 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, 11, 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, 249 and 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 some of them may preserve partial activity that can be promoter selective and temperature sensitive.16, 17 In addition, mutated p53 protein may exert a dominant negative effect on wild-type p53. Some p53 mutants may also gain new functions, which can contribute to cancerogenesis. Preliminary data show that the mutated p53 gain-of-function phenotype may be present also in CLL.18
Currently, there is not sufficient evidence to consider specific mutations or clone size in the diagnostic process, but this information should be assessed and recorded. It is important that mutations should be compared with currently available databases (www-p53.iarc.fr). If residual function is preserved and the mutation has rarely or never been reported in cancer, the results should be reassessed and the investigation repeated for confirmation, as technical artifacts can sometimes cause spurious results.
When to analyze 17p deletion and TP53 mutation
It is important to emphasize that current recommendations are based on retrospective analyses and subgroup analyses. No prospective trial data has shown superiority of one treatment regimen/concept for patients with TP53 mutations.
TP53 abnormalities should be assessed in:
All patients included in clinical trials.
Outside clinical trials in patients requiring therapy who would be eligible to an allogeneic stem cell transplantation or other intensive therapies (e.g., FCR and BR); TP53 abnormalities should be investigated immediately before treatment decision (results at diagnosis can change over time because of the clonal evolution).
Previously treated patients with wild-type TP53 at the time of treatment, should be retested when further therapy is needed and results can be expected to influence choice of therapy.
How to analyze TP53 status
The percentage of CLL cells in peripheral blood will influence the detection. Normally, in cases requiring therapy the majority of peripheral blood cells will belong to the CLL clone, but exceptions exist. If the proportion of leukemic cells in peripheral blood is lower than 50%, using bone marrow or lymph node tissue as source to investigate TP53 status may be considered especially when using direct Sanger sequencing. Alternatively, usage of CD19+ cells can be considered.
Detection of TP53 mutations
Although TP53 mutation may be present in a small proportion of leukemic cells, the presence of minor subclones is relatively rare (unpublished observations). Several methods based on analysis of genomic DNA, complementary DNA or RNA may be used, and each method has both advantages and shortcomings. For the purpose of this review, we will discuss these as well as minimum requirements to obtain meaningful information. In this regard, it is important to stress that for all techniques, mutations must be confirmed on a separate PCR reaction and Sanger sequencing.
(1) Direct Sanger sequencing of genomic DNA or complementary DNA: Usage of genomic DNA is preferable, because some mutations may lead to RNA degradation because of nonsense-mediated mRNA decay.19 Sequencing of (at least) exons 4–9 is recommended as the vast majority of mutations is located in this region (>95%).13 In a recent analysis of 268 TP53 mutations in CLL no mutations in exons 2, 3 and 11 were detected. Exon 10 harbored 4% of all TP53 mutations.13 Primers and reaction conditions can be found at www-p53.iarc.fr.
For centers planning TP53 mutation testing, direct sequencing can be considered as relatively simple approach, available at most laboratories. However, the sensitivity of direct sequencing is limited and therefore small subclones with TP53 mutation are unlikely to be detected. Sequencing analysis must be performed on forward and reverse reactions. Confirmation of mutations by a separate PCR reaction is mandatory.
Advantage: Provides direct information about the type of mutation.
Shortcomings: Relatively time consuming, low sensitivity (∼25% of mutated alleles in the sample).
(2) Pre-screening techniques such as denaturing high-performance liquid chromatography or single-strand conformation analysis have the advantage of higher sensitivity and lower costs. Abnormal screening results must be confirmed by Sanger sequencing in an independent PCR to exclude analytical artifacts.
Advantages: Fast, simple, economic and highly sensitive.
Note: Does not provide data on specific mutation (requires direct sequencing for confirmation).
(3) Functional analysis of separated alleles in yeast: The principle of this methodology is based on the cloning of complementary DNA from tumor samples into modified yeast cells. Gene expression triggers transcription of a reporter gene (ADE2—enzyme for adenine synthesis). Non-functional p53 leads to accumulation of a red intermediate product of adenine metabolism. Sequencing of templates from red yeast colonies bearing mutant TP53 for direct mutation identification is needed.
Advantages: Fast, cheap, no instrumentation required readout of transcriptional activity and sensitivity ∼10% of mutated DNA in the sample.
Shortcomings: May not pick up mutations leading to RNA degradation because of nonsense-mediated RNA decay, that is, some nonsense or frame-shift mutations.
Note: Underlying mutation should always be determined by sequencing (functional analysis of separated alleles in yeast methodology is not sufficient by itself).
(4) Arrays: Two platforms are currently in use (but not widely available); Affymetrix/Roche (GeneChip Arrays and p53 AmpliChip) and arrayed primer extension.20
The GeneChip arrays are based on short immobilized nucleotides with central base changes representing all potential nucleotide exchanges characterized by significantly different levels of hybridization to analyzed DNA. This approach was used for the first generation of p53 sequencing chips—the Affymetrix p53 GeneChip (Affymetrix, Santa Clara, CA, USA). Further development resulted in Roche p53 AmpliChip (Roche, Pleasanton, CA, USA), which was designed as a diagnostic tool for detection of all single base pair substitutions and single nucleotide deletions in exons 2–11 and splice sites and could reach a detection limit 1–2% of mutated DNA. Arrayed primer extension is based on incorporation of one of four labeled dideoxyNTPs into immobilized oligonucleotide primers. The TP53 arrayed primer extension array was designed to detect 95% of known TP53 mutations in exons 2–9.21
Advantages: Amplichip—fast, user friendly; Affymetrix custom GeneChip arrays—mutational analysis of multiple genes in parallel is feasible.
Shortcomings: Detects only mutations with probes printed on the array (Amplichip—all single base pair substitutions and single nucleotide deletions in exons 2–11, and splice sites). These assays are currently not widely available.
(5) Sequencing with next-generation technology: With the advent of high-throughput sequencing approaches, it is foreseeable that larger laboratories with high-case throughput will use this technology as a fast and cost effective approach. The discussion of the different technologies is beyond the scope of this review, but a number of next generation sequencing machines for diagnostic purposes are in use or under development.
Advantages: Very high and variable sensitivity based on coverage of analyzed sequences.
Shortcomings: Initial upfront cost of instrumentation, useful and economic only with high throughput of samples or target genes.
In addition to the detection of 17p deletion or TP53 mutation, there may be alternative lesions in the p53 pathway, which may contribute to the outcome of the disease.22, 23, 24 For the time being, functional defects of the p53 pathway beyond TP53 loss or mutation are a research topic and not considered for application in routine diagnostics. Although methods to detect the CLL cells’ response to DNA damage or p53 activation may be suitable surrogates of TP53 status (e.g., p53 target induction after DNA damage or interference of MDM2-p53 interaction), the current data is not sufficient to recommend the use of these techniques outside of the research scene.
Analysis, reporting and quality control of TP53 mutations
General recommendations for sequencing methods include the analysis of both forward and reverse strands to confidently identify or exclude TP53 mutations. Mutations should be confirmed in two separate PCR reactions because of the chances of polymerase errors leading to false positive results. This recommendation is highly dependent on the protocol used, as some proof-reading polymerases have significantly lower error rates than conventional Taq polymerases, and regional guidelines may differ from one country to another. It is strongly recommended that mutations that have not been previously characterized are confirmed in two independent reactions.
The report must include the parts of TP53 analyzed and the methodology used, with corresponding caveats regarding sensitivity and specificity of the method used. In cases where a TP53 mutation is found, this should be described according to the HGVS guidelines (http://www.hgvs.org/mutnomen) including both the nucleotide change at the DNA level as well as the amino-acid change at the protein level. The report should always include the reference TP53 sequence accession and version number used for the analysis, where nucleotide 1 is always the A of the ATG-translation initiation codon.
External quality assurance is a requisite of all accreditation bodies for molecular genetics; therefore, all laboratories should ensure that they participate in relevant external quality assurance or sample exchange programs. In the absence of national or international external quality assurance schemes for TP53 mutation analysis, it is recommended that all laboratories performing sequencing analysis are enrolled in a sequencing external quality assurance to ensure ongoing quality of data and interpretation (e.g., EMQN DNA-sequencing scheme, http://www.emqn.org/emqn/Schemes).
Irrespective of exchange programs, a simple assessment of test quality is the monitoring of the incidence of TP53 mutation in cases with deletion of 17p, which should be above 70%. Incidences below 60% should lead to reassessment of the technique's performance and procedures.
Clinical consequences derived from positive result
It is widely accepted that patients with TP53 abnormalities have an aggressive clinical course, require earlier intervention because of progressive disease and clinical symptoms, and respond poorly to therapy. This notion is based on data obtained in patients included in clinical trials, requiring therapy and thereby with poor prognosis. There are limited comprehensive studies of the natural history of CLL with TP53 abnormalities in unselected patients.
At diagnosis, the incidence of TP53 mutation has been reported to be 3.7%25 and it is important to stress that there are a subgroups of patients with 17p deletion (and mostly mutated IGHV status) who are not progressing for years.26, 27 As disease progresses, incidence rises to 10–12% (first line treatment) and ∼40% (F-refractory CLL).8
This emphasizes that treatment should only be initiated in the presence of active, symptomatic disease as recommended by the International workshop on CLL.28
For patients with TP53 mutation and treatment indication current guidelines are mainly based on outcome data of retrospective analyses from clinical trials and single center cohorts. Although the consequences of TP53 mutations as independent predictors of poor response and outcome are almost unequivocal, treatment recommendations will be based on cross-trial comparisons and not prospective randomized studies.
The authors hope that the careful but stringent integration of TP53 mutation analysis as recommended in this paper will help to unfold the clinical impact of TP53 abnormalities in CLL. Some recommendations are provided in Table 2.
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The authors declare no conflict of interest.
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Pospisilova, S., Gonzalez, D., Malcikova, J. et al. ERIC recommendations on TP53 mutation analysis in chronic lymphocytic leukemia. Leukemia 26, 1458–1461 (2012) doi:10.1038/leu.2012.25
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