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

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.


Specification
Variants in +/-2 intronic bases Rationale Most splice site variants in the TP53 gene lead to frameshift (see null variants).Only variants in the acceptor site of intron 3 (preceding exon 4) theoretically lead to inframe exon skipping, but various aberrantly spliced transcripts might be formed.Interpretation Always pathogenic (oncogenic)

Specification
Variants in +/-5 intronic bases Rationale Variants in nucleotides adjacent to canonical splice sites might affect splicing.The evidence can be found at the MutSpliceDB (https://brb.nci.nih.gov/splicing).For the TP53 gene, so far, only variants in position c.375+5 have documented an impact on splicing 23 , but a continuous extension of the data may be expected.Interpretation Variants with experimental evidence documenting splicing defect -likely pathogenic (oncogenic).

Specification
Variants in inner parts of introns further from splice sites and in 3' and 5'UTR regions Rationale Variants may affect transcription or splicing, but there is generally lack of evidence and large number of population variants are located in these regions.Interpretation Analysis is not recommended in routine practice.
Might be classified as likely pathogenic (oncogenic) if experimental evidence exists

Specification Deletions and insertions not disturbing the reading frame Rationale
Limited data on the functional impact exist.The vast majority of so-far analyzed inframe deletions within the DNA-binding domain showed impaired anti-proliferative capacity in H1299 cells 24 .The functional data are to be found via The TP53 database for some but not all tested variants.ERIC initiated the study of the impact of the inframe variants found in patients with CLL; all 45 tested variants showed disturbed functionality (manuscript in preparation).Interpretation Within the DNA-binding domain -likely pathogenic (oncogenic) Outside the DNA-binding domain -VUS unless functional data or proven association with Li-Fraumeni or hereditary cancer syndromes exist.

Synonymous variants
Specification Single-nucleotide change not leading to amino acid change Rationale Not all synonymous variants are silent.The variants present at the exon-intron boundary might affect splicing 25 (see Supplementary Figure 1).Care must be taken not to exclude all synonymous variants during the filtering in bioinformatics pipelines.Interpretation Most are benign but specific variants affecting splicing are pathogenic (oncogenic).

Specification
Single-nucleotide change variants leading to amino acid change Rationale Amino-acid change mostly affects p53 protein structure or DNA binding ability.Interpretation Variants with concordant data from functional studies 24,26,27 can be directly interpreted as (likely) pathogenic (oncogenic) -most variants, or (likely) benignminority of variants.For variants with discordant or lacking functional data further consideration is required (see Supplementary Figure 1).Caution: A few specific variants located in borderline exon/intron nucleotides affect splicing although they were classified as functional (see Supplementary Figure 1).Population variants: Several missense variants occur in a human population without affecting p53 function.Variant c.215C>G p.Pro72Arg is the most common benign SNP, others are listed in Supplementary Figure 1.The rare variants should be checked using Clingen repository 28 and GnomAD 22 .

Conclusion:
Example: A pathogenic variant was found within the TP53 gene.TP53 mutations are associated with adverse prognosis and poor response to chemoimmunotherapy in CLL and therefore such treatment should be avoided (PMID: 33091559 or other reference(s) of current national or international guidelines).

Or: No pathogenic variant within the TP53 gene was detected
The result should be interpreted with respect to the proportion of tumor cells in the primary sample and the separation method used.A low proportion of tumor cells in the sample may lead to a false negative result or a decreased VAF.

Mutational analysis of the TP53 gene
Patient name/id: * Date of sample collection: Date of birth: Date of sample delivery: Gender: Result issued: Reason for referral: Analytical method description (region sequenced, method description including bioinformatics pipeline): Minimal coverage of the target region: Detection limit of the method: Variants are described according the Human Genome Variation Society (HGVS) nomenclature Version xx.xx.

Variant interpretation:
Functional impact and pathogenicity of variants was assessed based on the following tools: The interpretation refers to the time of issuing of the report and may change in the future due to additional evidence.Validated polymorphisms and benign/likely benign variants are not included in this report and can be provided upon request.

Method limitations:
Example: The method cannot detect large duplications and deletions, and complex rearrangements within the tested regions of TP53 gene.The procedure cannot distinguish between somatic and germline variants without testing of normal tissue from the same individual.In the case of justified suspicion of the germinal origin of a variant with VAF>50% (young age, family history), the examination needs to be repeated from non-tumor DNA.
* Unique patient identification, the date of primary sample collection and the date of the issue of the report should be on each page of the report (in a header or a footer of the document).
ⱡ Co-validation and co-signature by a second competent person is recommended (and mandatory in some countries).
Note: Pages should be numbered in a format: 1/2, 2/2 Analysis performed by ⱡ : Name and function Result issued by: Name and function ⱡ

Supplementary Table S6. Details specifying the classification of TP53 variants detected in CLL patients. Truncating = null variants
SpecificationFrameshift insertions/deletions, nonsense variants Rationale Termination of translation due to the formation of a premature stop codon leads to non-functional protein.Transcripts bearing premature stop codons are often degraded via nonsense-mediated RNA decay resulting in decreased protein level Interpretation Always pathogenic (oncogenic) irrespective of their presence/absence in databases.
Template report form.Please check for the most updated version on www.ericll.org