ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-2024 update

Abstract

In chronic lymphocytic leukemia (CLL), analysis of TP53 aberrations (deletion and/or mutation) is a crucial part of treatment decision-making algorithms. Technological and treatment advances have resulted in the need for an update of the last recommendations for TP53 analysis in CLL, published by ERIC, the European Research Initiative on CLL, in 2018. Based on the current knowledge of the relevance of low-burden TP53-mutated clones, a specific variant allele frequency (VAF) cut-off for reporting TP53 mutations is no longer recommended, but instead, the need for thorough method validation by the reporting laboratory is emphasized. The result of TP53 analyses should always be interpreted within the context of available laboratory and clinical information, treatment indication, and therapeutic options. Methodological aspects of introducing next-generation sequencing (NGS) in routine practice are discussed with a focus on reliable detection of low-burden clones. Furthermore, potential interpretation challenges are presented, and a simplified algorithm for the classification of TP53 variants in CLL is provided, representing a consensus based on previously published guidelines. Finally, the reporting requirements are highlighted, including a template for clinical reports of TP53 aberrations. These recommendations are intended to assist diagnosticians in the correct assessment of TP53 mutation status, but also physicians in the appropriate understanding of the lab reports, thus decreasing the risk of misinterpretation and incorrect management of patients in routine practice whilst also leading to improved stratification of patients with CLL in clinical trials.

Fig. 1. Breakdown of TP53 aberrations detected in CLL based on the presence of TP53 mutations, their allele burden, and concomitant del(17p) as assessed by FISH.

Values were adopted from published studies employing ultra-deep NGS to detect TP53 mutations [–10, 66]. High VAF—variants >10% VAF, low VAF—variants 1–10% VAF, except for two studies where variants <1% and >1% could not be distinguished [4, 5]. In patients with high VAF TP53 mutations, co-existence of del(17p) prevails. * In patients carrying low VAF TP53 mutation concomitant del(17p) is detected in only a minority of cases, but the true status is unknown due to the higher detection limit of FISH (>5% aberrant nuclei). The breakdown depicted here corresponds to pre-treatment cohorts (diagnosis or before frontline treatment). In the chemo-pretreated cohorts the proportion of patients with TP53 defects can reach 40% [1, 20, 106].

Fig. 2. Illustrative example of clonal distribution of TP53 variants.

The distribution of variant allele frequencies (%VAF; y-axis) of TP53 mutations detected in patients with CLL (x-axis). Variants present in the whole cancer population are clonal, otherwise, they are deemed subclonal. Variants <10% VAF are considered low burden. This distribution is valid when the sample contains >90% tumor cells. In samples with a low CLL cell fraction, a low VAF may, in reality, correspond to a clonal mutation.

Fig. 3. Responsibilities and cooperation between the laboratory and the physician with respect to TP53 mutation diagnostics and interpretation.

The laboratory is responsible for issuing the correct result and reports all pathogenic TP53 variants above the validated LoD. The result should be interpreted in the context of tumor cell content, separation method, and disease status. The physician decides about the treatment based on all available information: the laboratory results, the clinical characteristics of the patient, patient preferences, and the availability of the treatment.

Fig. 4. Spectrum of TP53 defects detected in CLL.

TP53 variant profile based on data collected for CLL patients in the UMD database; common polymorphisms have been omitted [82]. A Codon distribution with hot-spot variants depicted. Variants in codons 175, 248, and 273 are general hot spots, while the truncating frameshift variant in codon 209 is CLL-specific. B Exon distribution showing the prevalence of variants in exons 5–8. C Proportion of variant types out of all variants. D Proportion of variant types in individual domains. In the DNA-binding domain, missense variants prevail; conversely, truncating variants are predominant in the carboxy and amino termini.

Fig. 5. Classification of TP53 variants detected in CLL.

A classification algorithm showing the basic principles of assigning variants into pathogenicity/oncogenicity classes. A detailed version of the algorithm listing assistive tools and specific variants classified into respective categories can be found in Supplementary Figure 1. Databases instrumental in the interpretation of TP53 variants are listed in Supplementary Table S5. # Might be misclassified as synonymous or missense and listed as such in some databases. *Oncogenicity classification according to Horak et al. [90] is also acceptable. Occurrence according to the UMD database [82]. VUS variant of unknown significance.

Fig. 6. TP53 variants detected in CLL with respect to their type and interpretation.

Illustrative example based on data published in Malcikova et al. [6]. Common population variants have been excluded. A Breakdown based on assignment using proposed classification algorithm (color coding corresponds to Fig. 5). Concordant functional/non-functional: assessed by functional tests (Kato et al. [95], Giacomelli et al. [96] and Kotler et al. [97]). B Proportion of TP53 variants detected in CLL assigned to pathogenicity categories. VUS variant of unknown significance.