Eric recommendations for tp53 mutation analysis in chronic lymphocytic leukemia—update on methodological approaches and results interpretation

ABSTRACT In chronic lymphocytic leukemia (CLL), _TP53_ gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the _TP53_ gene, are associated with resistance to

chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of _TP53_ aberrations has been incorporated into routine clinical diagnostics to improve patient

stratification and optimize therapeutic decisions. The predictive implications of _TP53_ aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of

B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with _TP53_ defects. In this report, the _TP53_ Network of the European Research

Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for _TP53_ mutation analysis. Moreover, it provides guidance to ensure

that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since

next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of _TP53_ mutation

data generated by this methodology. SIMILAR CONTENT BEING VIEWED BY OTHERS ERIC RECOMMENDATIONS FOR _TP53_ MUTATION ANALYSIS IN CHRONIC LYMPHOCYTIC LEUKEMIA—2024 UPDATE Article Open access

16 May 2024 TIME TO FIRST TREATMENT AND P53 DYSFUNCTION IN CHRONIC LYMPHOCYTIC LEUKAEMIA: RESULTS OF THE O-CLL1 STUDY IN EARLY STAGE PATIENTS Article Open access 28 October 2020 CLONAL

EVOLUTION AND CLINICAL IMPLICATIONS OF GENETIC ABNORMALITIES IN BLASTIC TRANSFORMATION OF CHRONIC MYELOID LEUKAEMIA Article Open access 14 May 2021 INTRODUCTION Chronic lymphocytic leukemia

(CLL) displays a very heterogeneous clinical behavior, therefore prognostic and predictive markers play an important role in disease management. To date, the key decision-making biomarkers

in CLL are _TP53_ gene defects: chromosomal aberrations of 17p13, in particular deletions spanning the _TP53_ locus, and _TP53_ gene mutations, both of which are associated with adverse

disease outcome due to resistance to chemoimmunotherapy [1,2,3,4]. Early studies utilizing fluorescent in situ hybridization (FISH), for the detection of cytogenetic aberrations, revealed

that CLL patients carrying del(17p) have a significantly shorter overall survival compared to patients harboring other recurrent cytogenetic abnormalities, i.e., del(11q), trisomy 12, or

del(13q) [5]. Inactivation of the _TP53_ locus due to del(17p) is frequently associated with mutation(s) on the second _TP53_ allele. However, _TP53_ mutations also occur in the absence of

del(17p) in about 5% of untreated patients and are associated with a poor outcome, similar to the disease course observed in del(17p) CLL patients [6, 7]. More specifically, approximately

90% of patients with del(17p) carry a _TP53_ mutation; conversely, only 60–70% of patients with _TP53_ mutation also harbor del(17p), as detected by FISH [8,9,10,11,12]. The clinical utility

of _TP53_ mutation analysis in CLL has been well documented by many studies [7,8,9, 11, 13], including findings from prospective clinical trials [6, 14, 15] clearly showing that patients

carrying _TP53_ defects are resistant to chemoimmunotherapy. In this context, the advent of novel treatment options inhibiting B-cell signaling and anti-apoptotic BCL2 that proved

efficacious in patients harboring _TP53_ gene disruption [16,17,18] has brought an urgent need for accurate assessment of the _TP53_ gene status in routine clinical practice with the aim of

identifying those patients who would not benefit from chemoimmunotherapy, and hence should be considered for targeted agents. _TP53_ gene assessment should always be performed prior to

initiation of the first and every subsequent line of treatment [19]. That said, a few situations exist where _TP53_ mutational analysis may not be required, e.g., when the use of

p53-independent drugs is not possible due to either patient fitness or limited market access, or when the presence of a _TP53_ alteration has already been documented. The recent introduction

of high-throughput next-generation sequencing (NGS) has led to the identification of _TP53_ mutations with a low variant allelic frequency (VAF)—usually below the detection limit of

conventional Sanger sequencing—that may be positively selected with the use of chemotherapy, ultimately leading to the expansion of an initially minor _TP53_ mutant subclone into a prevalent

refractory clone [20,21,22,23,24]. Taken together, the recent therapeutic and technological advances necessitate an update of the previously published ERIC recommendations for _TP53_

mutation analysis in CLL [19], including assessment of the current methodological approaches as well as recommendations for the interpretation of the findings and the accurate reporting of

results. An overview of the updated recommendations is provided in Table 1. PROCEDURE DESCRIPTION MATERIAL FOR _TP53_ MUTATION ANALYSIS For most CLL patients, peripheral blood (PB) is an

appropriate starting material for _TP53_ mutation analysis. Nevertheless, an important factor influencing the result is the cancer cell fraction (CCF), and this is particularly relevant in

cases with a low lymphocyte count (<10 × 109/L and/or <60–70% lymphocytes in PB). This is usually evidenced in patients with predominant lymphadenopathy and few circulating clonal

cells, i.e., small lymphocytic lymphoma. In such cases, material enriched with tumor cells such as bone marrow (BM) or lymph node biopsies may be an alternative option. PB or BM should be

collected in tubes containing an anticoagulant, such as EDTA or heparin, followed by mononuclear cell separation by density gradient centrifugation to enrich the lymphocyte fraction. The use

of mononuclear cells might be insufficient when the specimen analyzed contains less than 60–70% lymphocytes and could lead to a false-negative result when using Sanger sequencing

(Supplementary Fig. S1). In such instances, selection of CD19+ cells using enrichment techniques such as RosetteSep or MACS should be performed to yield a higher CCF. Alternatively,

ultra-deep NGS, which has a much greater sensitivity level, can be performed and the VAFs corrected with respect to the CCF. Regarding tissue material, fresh/frozen material is strongly

preferred. Formalin-fixed, paraffin-embedded (FFPE) tissues are recommended only when no alternative sample is available as the fixation and embedding processes may hamper the analysis,

since: (i) FFPE material often contains highly degraded DNA fragments, therefore shorter amplicons are required for sequencing; (ii) the process of tissue fixation damages DNA through

cross-linking, thus reducing the number of intact DNA molecules added into the PCR [25]; and (iii) DNA can be chemically modified, leading to artefactual sequencing results (particularly

deamination and oxidation artifacts) [26,27,28]. Therefore, any variants detected in DNA samples from FFPE material should be confirmed by independent PCR and carefully verified using the

recommended databases (described below) before interpreting and reporting them as mutations. Finally, when considering the type of nucleic acid to analyze, genomic DNA is highly recommended.

Analyzing RNA may result in truncating or splice site variants being missed due to nonsense-mediated RNA decay [29]. In addition, using whole-genome amplification for diagnostic purposes is

discouraged as it may introduce a bias in allelic frequencies and could lead to allelic drop-out. REGION OF INTEREST At a minimum, the sequenced region of the _TP53_ gene must include exons

4–10, which corresponds to the DNA-binding domain (codons 100–300) and the oligomerization domain (codons 323–356). Sequencing of exon 10 is recommended as the frequency of mutations in

exons 9 and 10 is similar or even higher in exon 10 as documented by the recent studies [30] (Fig. 1). Optimally, exons 2–11 should be analyzed to cover the entire coding region [30]. _TP53_

gene profiling studies by NGS, which usually involves also exons 2, 3, and 11, have shown that variants can also occur in these exons, although their frequency is low (T. Soussi,

unpublished results; Fig. 1). As each exon is surrounded by a splice donor and a splice acceptor site, sequencing of +2/−2 intronic nucleotides is required to detect variants which may

impair splicing and translate to inactive proteins. SANGER SEQUENCING Primer sequences, as well as the protocol for performing the PCR, are available on the International Agency for Research

on Cancer (IARC) _TP53_ website (http://p53.iarc.fr/ProtocolsAndTools.aspx). This PCR protocol is adaptable and can be modified based on local experience. Bidirectional sequencing analysis

is the only acceptable strategy, and the chromatograms generated by Sanger sequencing should be carefully scrutinized to ensure that somatic variants present at lower allelic frequencies are

not overlooked; adjusting software settings to detect germline homozygous and heterozygous variants is not sufficient. The ERIC _TP53_ Network provides the opportunity to analyze Sanger

sequencing data via a web-based tool called GLASS [31]. This software was purpose-built to assist with the assessment of somatic gene variations and provides a standardized variant output as

recommended by the Human Genome Variation Society (HGVS). GLASS was specifically developed to support ERIC _TP53_ Network activities and is freely accessible at

http://bat.infspire.org/genomepd/glass/ or via the ERIC website (http://www.ericll.org/guidance-toolstp53/). Finally, although the relevance of pre-screening methods, such as denaturing

high-performance liquid chromatography and high-resolution melting analysis is decreasing, they remain a viable and cost-effective option. That notwithstanding, in order to identify the

specific variant, aberrant screening results must always be confirmed by Sanger sequencing in an independent PCR. NEXT-GENERATION SEQUENCING Targeted NGS can be used for the analysis of the

_TP53_ gene as a standalone assay or as part of a gene panel investigating several genes. Numerous commercially available ready-to-use analytical kits include the _TP53_ gene, and ERIC is

conducting a multi-center collaborative effort to assess and compare various pre-designed and custom gene panel technologies. Previous studies exploring the inter-reproducibility of targeted

NGS and Sanger sequencing for _TP53_ analysis demonstrated very good correlation of the results, specifically showing that all variants detected by Sanger sequencing are also detectable by

NGS [22, 23, 32,33,34,35]. A recent study also showed an excellent correlation between the results obtained from two different NGS platforms, namely, the Ion PGM (ThermoFisher) and the MiSeq

(Illumina) [33]. In addition, NGS is capable of detecting variants below the sensitivity threshold of Sanger sequencing, even VAFs as low as <1% [20, 22, 23]. Due to the low detection

limit of NGS, multiple subclonal mutations within the _TP53_ gene (i.e., convergent mutations) may be detected in some patients [20, 35]. To ensure the maximum applicability and reliability

of NGS, several important issues need to be addressed when establishing the methodology, as erroneous results can arise for various reasons (Table 2). DNA INPUT AND QUALITY Low input and/or

degraded DNA may result in false-negative results due to a sampling effect, and may also produce false-positive results as amplified errors might constitute a significant proportion of the

final sequencing library [36]. The initial amount of DNA should always be calculated with respect to the required limit of detection (LOD), keeping in mind that a human cell (two alleles)

contains approximately 6 pg of DNA. For reliable detection, the DNA input must ensure that the sample contains a sufficient number of variant molecules and that the variants can be

distinguished from background noise. For instance, at least 10 ng corresponding to approximately 1500 cells or 3000 alleles should be used to detect variants present at 1% VAF. This is also

relevant for techniques which require the starting amount of DNA to be distributed amongst individual nano-scale PCRs, e.g., the Fluidigm Access Array, RainDance Technology, or Wafergen.

Although DNA isolated from PB and BM is usually of good quality, testing the integrity of the DNA by agarose electrophoresis or specialized automated electrophoresis devices is recommended

(and often required) for NGS. Special attention is required when considering the quality and quantity of DNA obtained from FFPE samples due to the increased risk of false-positive as well as

false-negative results. LIBRARY PREPARATION Both amplicon-based and capture-based approaches are applicable. From a practical perspective, amplicon-based library preparations require much

smaller quantities of input DNA and the workflow tends to be simpler and less time-intensive and labor-intensive compared to capture-based methodologies. On the other hand, hybridization

capture-based approaches demonstrate better uniformity of coverage and generate fewer false-negative as well as false-positive calls as compared to amplicon-based techniques. When designing

in-house primers for amplicon-based libraries, it is important to check the primer positions against potential single-nucleotide polymorphisms (SNP) and ensure that the primers can

efficiently read across splice junctions. In order to establish an NGS assay with high detection sensitivity, proofreading polymerases with low error-rates are recommended. Incorporating

unique molecular identifiers into the library preparation helps to distinguish errors introduced artificially during the process from true low-frequency variants and also allows for more

accurate quantification (especially with PCR-based protocols) [37, 38]. Additional benchmarking studies are required to establish standard analytical methods that must then be checked for

accuracy and reproducibility. SEQUENCING AND COVERAGE The required coverage should be set to ensure that the call is statistically above the background noise. Generally, the minimal coverage

should not be less than 100 at any position within the regions of interest and the number of variant reads for reliable variant calling should be at least 10. The frequently reported mean

or median coverage of a diagnostic panel is non-informative as uncovered regions cannot be deduced from this average value and therefore a ≥99% minimum coverage percentage is a vital

requirement. Of note, the number of reads does not necessarily reflect the actual number of unique template gDNA molecules, as many reads will be duplicates generated during PCR

amplification. When employing longer reads, a confident overlap (>60–70%) between the paired reads is recommended in order to avoid the introduction of false-positive results. Calling

variants found in unbalanced regions with forward-reverse ratios of less than 10% (i.e., strand bias) should be avoided. DATA ANALYSIS Multiple commercial, as well as free, software tools

are available to analyze NGS data and, as the bioinformatics field is continuously evolving, no single tool is currently preferentially recommended. That said, it is of utmost importance to

use a pipeline that has been optimized, and validated, for the detection of low abundance variants that must be distinguished from background error noise. Another issue concerns the accurate

identification of insertions and deletions (indels), which may be missed during the alignment process, especially in the case of complex indels. Numerous indel-calling tools have been

developed that often vary in the manner by which they detect indel breakpoints. Performance evaluations of indel-calling software have revealed limitations in detection; consequently, manual

inspection of the data is always recommended and is particularly required for indel variants and variants close to the detection limit. LIMIT OF DETECTION LOD refers to the lowest VAF that

is reproducibly detectable by the particular method under specific well-defined conditions. The LOD is a function of both the initial DNA input and the coverage achieved. The NGS assay

should be established, and validated, to at least reliably identify variants detectable by Sanger sequencing and avoid false-positive calls with VAF above the Sanger sequencing detection

limit (e.g., minimum LOD is 10% VAF). LOD should be set by taking into account non-uniformity of coverage across the analyzed sequence and an inconsistent error distribution. The occurrence

of sequencing errors varies depending on the nucleotide position and composition and is also platform-dependent, with C:G>T:A being the most frequent using Illumina platforms [39]. The

error rate is also influenced by the specific sequence context (e.g., homopolymers are more prone to erroneous variant calling). The issue of detection limit and how it can influence the

interpretation of findings is discussed in the following section. CLINICAL REPORTING AND INTERPRETATION OF THE RESULTS VARIANT DESCRIPTION Detected variants should be described using the

nomenclature devised by the HGVS nomenclature (http://varnomen.hgvs.org/) [40]. Several software programs are available to ensure adherence to standardized nomenclature (e.g., Mutalyzer;

https://www.mutalyzer.nl/). Variants should be described at both cDNA and protein level, and the reference sequence number and version including the transcript and protein variant should be

stated (see Supplementary Material). To standardize the output, the preferred coding DNA reference sequence is the stable Locus Reference Genomic sequence (LRG;

http://ftp.ebi.ac.uk/pub/databases/lrgex/LRG_321.xml) [30]. Transcript and protein variants 1 should be used (LRG_321t1, LRG_321p1). Special attention is warranted when annotating variants

detected by NGS, especially since many bioinformatics pipelines do not fulfill the requirements for correct variant description according to the HGVS nomenclature. More specifically: (i)

insertions and deletions are often not handled accurately; (ii) duplications are often misinterpreted as insertions; (iii) varying reference sequences for _TP53_ within the same output are

used; and (iv) the 3′ rule is not always implemented correctly. This is of particular importance for _TP53_ and other genes that are oriented in the reverse direction on the chromosome. In

such situations, the alignment and variant calling steps may introduce errors if aligning to the 3′ end with respect to the chromosome position rather than the coding sequence orientation.

INTERPRETATION DATABASES The detected variant should be checked using locus-specific databases, i.e., either the IARC _TP53_ database (http://p53.iarc.fr/TP53GeneVariations.aspx) [41] or the

_TP53_ website (UMD database; http://p53.fr/) [42]. These databases compile data from peer-reviewed literature as well as general databases, and provide information about: (i) the

functional impact of all possible single-nucleotide exchanges within the coding region; (ii) the variant frequencies noted in both the somatic and the germline context; and (iii) additional

relevant information, including links to other resources. The _TP53_ website also provides a web-service tool called Seshat that is capable of managing files generated from NGS both in the

vcf and bam formats. Seshat helps the user to: (i) check the variant nomenclature for consistency and generate a full description of each variant formatted according to HGVS; (ii) assess the

pathogenicity of each variant according to general prediction algorithms and algorithms developed specifically for analyzing the _TP53_ gene; and (iii) obtain functional and structural data

for each _TP53_ variant. Finally, variants can also be checked using the COSMIC (http://cancer.sanger.ac.uk/cosmic) or ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) databases; however,

these databases are only recommended as a complementary analysis to the locus-specific databases. POLYMORPHISMS AND NEUTRAL VARIANTS In general, it is not recommended to include common

polymorphisms and benign variants in the report to physicians. If, however, the local practice requires that these variants are detailed in the clinical report, it should be clearly

indicated that the detected variant is not clinically relevant. According to the IARC database, there are six validated exonic polymorphisms within the _TP53_ gene; two are synonymous

(c.108G>A: p.Pro36= and c.639A>G: p.Arg213=) and four are nonsynonymous (c.91G>A: p.Val31Ile; c.139C>T: p.Pro47Ser; c.215C>G: p.Pro72Arg, and c.1096T>G: p.Ser366Ala). The

most frequent polymorphism is c.215C>G: p.Pro72Arg, where the ancestral allele C coding for proline is less frequent in the general population than the allele G [43] with

latitude-dependent variations. Although the two alleles were reported to have different capabilities in inducing apoptosis and G1 arrest [44], studies analyzing the clinical impact of

p.Pro72Arg and its associations with _TP53_ mutations in CLL reported inconclusive results [45,46,47,48]. Reporting of the p.Pro72Arg status is therefore not recommended due to a lack of

convincing evidence with regard to prognostic or clinical relevance. Using dbSNP for filtering out polymorphisms and neutral variants is strongly discouraged as many variants listed in dbSNP

exhibit loss of function and are frequently observed in cancer patients despite not being reported as pathogenic in ClinVar [49]. More specifically, of the 100 most frequent deleterious

somatic variants described in the IARC database, 65 are present in dbSNP147 and only 34 are described as being pathogenic [41]. Using the data set collected within the context of the Genome

Aggregation Database (gnomAD) is more accurate; however, it should be noted that several pathological variants are also listed in this database (http://gnomad.broadinstitute.org/, originally

Exome Aggregation Consortium [43]). VARIANTS WITH PRESERVED ACTIVITY If a rare variant or a variant with preserved functionality is detected, it is recommended to repeat the entire

analysis, starting from the PCR step, so as to exclude analytical errors. If the variant is verified and the VAF is approximately 50%, suggesting a germline origin, it is advisable to verify

the germline or somatic nature of the variant by testing patient-matched germline DNA, obtained from CD3+ cells, saliva, a buccal swab or a skin biopsy (it is advised to rule out the

contamination with CLL cells by flow cytometry or by testing the patient-specific IGHV rearrangement). Variants that have preserved transactivation capabilities are often found as germline

and the carriers do not show any personal or family cancer-history associated with Li-Fraumeni or another cancer-predisposing syndrome. Specific examples of variants that should be

considered with caution and are often inaccurately reported are c.704A>G: p.Asn235Ser or c.847C>T: p.Arg283Cys. If the somatic origin of such a variant is confirmed, the variant should

be reported to the clinician clearly stating that a variant of unknown significance was found. In the case that the variant is of germline origin, reporting should follow the

recommendations of The American College of Medical Genetics and Genomics [50, 51] (recommendations of The European Society of Human Genetics are currently under preparation). INTRONIC

VARIANTS Variants affecting splice sites (+2/−2 intronic nucleotides) are considered pathogenic as they lead to aberrant mRNA splicing. Pathogenicity of intronic variants outside the donor

and acceptor sequence is largely unexplored, and therefore they should not be reported unless their functional impact is proven at the RNA or protein level by documenting the presence of

aberrantly spliced transcripts or shortened protein products. As these methods are not usually accessible in diagnostic labs, reporting of intronic variants with the exception of splice

sites is not recommended within clinical routine. SYNONYMOUS VARIANTS If a synonymous variant is detected, it is important to check its predicted effect on splicing [52] via the IARC

database or the _TP53_ website. For instance, synonymous variants in codon 125 (c.375G>A and c.375G>T) have been found in various cancers and Li-Fraumeni families and shown to affect

the splicing of exon 4 [53], therefore they are classified as pathogenic. INDEL VARIANTS Insertions and deletions leading to the formation of a premature stop codon (frameshift variants) as

well as in-frame indels within the DNA-binding domain are considered as likely pathogenic. CLINICAL REPORTING OF SUBCLONAL VARIANTS WITH LOW VARIANT ALLELE FREQUENCY DETECTED BY NGS The

definition of the term “subclonal” is generally used to describe variants that are not present in the entire tumor population, as opposed to “clonal” [21]. Terms such as “minor subclone”,

“low-burden”, or “low-level” variants refer to variants with allelic burdens below the detection limit of Sanger sequencing, i.e., <10% VAF. Of note, caution is necessary when

interpreting VAFs as its calculation does not take into consideration the CCF and the presence of genomic copy number aberrations. Therefore, it is important to bear in mind that a 5% VAF

could be clonal if the CCF is only 10% and no del(17p) or copy-neutral loss of heterozygosity is present. Several publications have suggested that _TP53_ mutations within minor clones are

clinically relevant, which is particularly important considering that administration of therapeutic regimens based on DNA-damaging agents represents a risk for the selection of these

low-level _TP53_-mutated subclones [20,21,22,23, 33, 54]. However, the extent of the risk posed by minor subclones harboring _TP53_ mutations has not been conclusively defined, and the

current evidence on the poor outcome of _TP53_-mutated patients treated with chemoimmunotherapy in clinical trials is based on data obtained using Sanger sequencing only. Therefore,

currently, the presence of minor subclonal mutations should not impact clinical decision-making. Based on current knowledge, the recommended threshold for reporting of mutations detected by

NGS should reflect the Sanger-like threshold of approximately ~10% VAF. That said, bearing in mind that the 10% threshold is arbitrary, variants with 5–10% VAF can also be reported; however,

always mentioning in the report that the clinical significance of _TP53_ mutations with VAF 5–10% is currently unknown, since we are lacking data from prospective clinical studies

addressing this issue. Importantly, NGS technology should be validated to a LOD above which there are no false positives (minimum 10% VAF). Confirmation of mutations detected at the level

near the validated LOD is desirable either by Sanger sequencing or, in the case of minor clone variants, by digital PCR, independent NGS run or allele-specific PCR. REPORT FORM In addition

to the obligatory standard medical report content (e.g., patient and lab identifiers, date of sampling, type of material), the report should always contain the following information: (i) the

type of analysis and description of the method: methodology used, exons analyzed, LOD, and coverage in the case of NGS (median and ≥99% minimum); (ii) results and interpretation:

description of the identified variant(s) according to the HGVS nomenclature, reference sequence used, type of variant (missense/truncating etc.), effect according to the _TP53_

locus-specific database, frequency, and any known association with cancer; (iii) conclusion: clinical consequence of the variant and summary of the finding in the context of the current

knowledge; and (iv) other optional data: VAF of the detected variant if available (estimations from Sanger sequence traces can also be informative), comparison with a previously tested

sample from the same patient and, if evidenced, description of clonal evolution. All labs issuing clinical reports of their results must have accreditation according to their national

authorities. ERIC is also regularly conducting _TP53_ mutational Analysis Certification to confirm the reliability and reproducibility of the results provided by participating labs. Examples

of report forms for both Sanger sequencing and NGS are provided in the Supplementary Material and a template report form can be found on the ERIC website (http://www.ericll.org/).

PUBLISHING AND SCIENTIFIC REPORTING IN THE DATABASES It is important to distinguish between clinical reporting and reporting variants for research purposes in scientific journals. Data from

publications are transferred to databases, and these databases then serve as the source of information for general use [42, 49]. For this reason, in order to prevent incorrect entries, it is

essential to follow specific rules in addition to all above-mentioned basic procedures: (i) using consistent sample and patient identifiers if the data are repeatedly published, as

inconsistent identification leads to redundancy in mutation databases; (ii) including the genomic coordinate and reference genome in the variant description to avoid ambiguities; (iii)

listing all variants that are found in the patient including synonymous and other benign variants [55]. It is recommended to include the complete list of variants in the Supplementary

Material, with appropriate description of their clinical significance. Note that if more than one variant in a patient is found, all variants should be listed. Centers following ERIC

recommendations are kindly asked to mention ERIC in the “Material and methods” section of their studies and refer to this manuscript. CONCLUDING REMARKS In CLL, inactivation of the _TP53_

gene by deletion and/or mutation is strongly associated with adverse prognosis and refractoriness to chemoimmunotherapy. Detection of del(17p) and _TP53_ gene mutations has become an

integral part in routine diagnostics and should always be performed before deciding about treatment. Analysis of _TP53_ exons 4–10 is a minimal requirement; however, ideally, the entire

coding sequence, i.e., exons 2–11, should be analyzed, and this can be performed by either bidirectional Sanger sequencing or NGS. NGS also allows the parallel analysis of multiple genes and

is capable of identifying variants undetectable by Sanger sequencing. That notwithstanding, NGS currently faces certain technical limitations and may lead to problems with data

interpretation. The clinical importance of mutations within minor clones remains an unresolved issue and there is currently not enough evidence for making therapeutic decisions based on the

presence of mutations undetectable by Sanger sequencing. To assist the community with the implementation of _TP53_ mutational analysis in a harmonized manner, ERIC created the _TP53_ Network

with the following objectives: regular certification of laboratories for _TP53_ mutation status assessment (both for Sanger and NGS), the organization of educational events, and regular

updating of recommendations for _TP53_ analysis. The Network also provides tools facilitating laboratories to achieve reliable and comparable results that are accessible via the ERIC web

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publications. Hum Mutat. 2017;38:339–42. Article  CAS  Google Scholar  Download references FUNDING Supported by the IMI 2 HARMONY JU under GA No 116026, this JU receives support from the

EU’s H2020 R&I program and EFPIA. Further supported by the EU Horizon 2020 projects MEDGENET 692298, AEGLE 644906, projects CEITEC 2020 (LQ1601), NCMG research infrastructure (LM2015091

funded by MEYS CR), project FNBr 65269705, FM MU ROZV/24/LF/2016, DFG (SFB1074, projects B1 and B2, and EU (FIRE CLL)), and the Swedish Cancer Society and the Swedish Research Council.

Publication reflects only the authors’ views and the Commission is not responsible for any use that may be made of the information it contains. AUTHOR INFORMATION Author notes * J.

Malcikova, E. Tausch, and D. Rossi contributed equally to this work * P. Ghia and S. Pospisilova contributed equally to this work. AUTHORS AND AFFILIATIONS * Department of Internal Medicine

— Hematology and Oncology, University Hospital Brno and Medical Faculty, Masaryk University, Brno, Czech Republic J. Malcikova & S. Pospisilova * Central European Institute of

Technology, Masaryk University, Brno, Czech Republic J. Malcikova & S. Pospisilova * Department of Internal Medicine III, Ulm University, Ulm, Germany E. Tausch & S. Stilgenbauer *

Hematology, Oncology Institute of Southern Switzerland, Institute of Oncology Research, Bellinzona, Switzerland D. Rossi * Department of Immunology, Genetics and Pathology, Science for Life

Laboratory, Uppsala University, Uppsala, Sweden L. A. Sutton & R. Rosenquist * Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden L. A. Sutton & 

R. Rosenquist * Université Pierre et Marie Curie, Paris, France T. Soussi * INSERM, U1138, Centre de Recherche des Cordeliers, Paris, France T. Soussi * Department of Oncology-Pathology,

Karolinska Institutet, Cancer Center Karolinska, Stockholm, Sweden T. Soussi * Division of Hematology, University Hospital Zürich, University of Zürich, Zürich, Switzerland T. Zenz *

Department of Hematology, Academic Medical Center, Amsterdam, The Netherlands A. P. Kater * Department of Hematology, Rigshospitalet, Copenhagen, Denmark C. U. Niemann * Centre for Cancer

Research and Cell Biology, Queen’s University Belfast, Belfast, UK D. Gonzalez * Department of Hematology, Hôpital Pitié-Salpêtière, AP-HP, Sorbonne Universités-UPMC University, Paris,

France F. Davi * Centro de Investigación del Cancer and Centro de Investigación Biomédica en Red de Cáncer (CIBERONC), University of Salamanca, Salamanca, Spain M. Gonzalez Diaz * Department

of Haematology, Hospital de la Santa Creu I Sant Pau, Autonomous University of Barcelona, Barcelona, Spain C. Moreno * Division of Haematology, Department of Translational Medicine,

University of Eastern Piedmont, Novara, Italy G. Gaidano * Institute of Applied Biosciences, CERTH, Thessaloniki, Greece K. Stamatopoulos * Division of Experimental Oncology, IRCCS San

Raffaele Scientific Institute, Università Vita-Salute San Raffaele, Milan, Italy P. Ghia Authors * J. Malcikova View author publications You can also search for this author inPubMed Google

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CONSORTIA ON BEHALF OF THE EUROPEAN RESEARCH INITIATIVE ON CHRONIC LYMPHOCYTIC LEUKEMIA (ERIC) — TP53 NETWORK CORRESPONDING AUTHORS Correspondence to P. Ghia or S. Pospisilova. ETHICS

DECLARATIONS CONFLICT OF INTEREST JM and SP: consultancy fees and travel grants from Gilead and Abbvie. DR: research funding from Abbvie and Gilead, consultancy fees from Abbvie, Janssen,

Gilead. LAS: honoraria for consultancy from Gilead and Janssen. TZ: honoraria from Janssen, Gilead, Abbvie, Vaniam Group, Roche. APK: research funding from Janssen, Gilead, Abbvie, Celgene,

Roche. CN: research funding from Novo Nordisk Foundation, Danish Cancer Foundation and Abbvie and consultancy fees and/or travel grants from Roche, Janssen, Novartis, Gilead, and Abbvie. FD:

consultant fees from Gilead. CM: consultant fees from Janssen, Gilead, Pharmacyclics and research funding from Roche and Gilead. GG: consultancy fees from Janssen, Gilead, Roche, Morphosys,

and Abbvie. KS: research support from Janssen Pharmaceuticals, Gilead Sciences, Novartis SA, and Abbvie. RR: consultancy fees from Gilead and Roche. SS: honoraria for consultancy, honoraria

and research grants from AbbVie, Celgene, Genentech, Gilead, GSK, Hoffmann La-Roche, Janssen, Novartis, Pharmacyclics. PG: honoraria for consultancy and research grants from AbbVie,

Janssen, Gilead, Roche. The remaining authors declare that they have no conflict of interest. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY FIGURE 1 – ILLUSTRATIVE EXAMPLE OF A CLL PATIENT

WITH A LOW LYMPHOCYTE COUNT IN THE PERIPHERAL BLOOD. SUPPLEMENTARY FILE 1 – EXAMPLE OF A REPORT FORM: NEGATIVE RESULT, SANGER SEQUENCING SUPPLEMENTARY FILE 2 – EXAMPLE OF A REPORT FORM:

NEGATIVE RESULT, NGS SUPPLEMENTARY FILE 3 – EXAMPLE OF A REPORT FORM: HOT SPOT MUTATION, NGS SUPPLEMENTARY FILE 4 – EXAMPLE OF A REPORT FORM: 2 MUTATIONS (SPLICE SITE AND DELETION), SANGER

SEQUENCING SUPPLEMENTARY FILE 5 – EXAMPLE OF A REPORT FORM: NONSENSE MUTATION WITH LOW VAF, NGS SUPPLEMENTARY FILE 6 – EXAMPLE OF A REPORT FORM, VARIANT OF UNKNOWN SIGNIFICANCE, SANGER

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ARTICLE CITE THIS ARTICLE Malcikova, J., Tausch, E., Rossi, D. _et al._ ERIC recommendations for _TP53_ mutation analysis in chronic lymphocytic leukemia—update on methodological approaches

and results interpretation. _Leukemia_ 32, 1070–1080 (2018). https://doi.org/10.1038/s41375-017-0007-7 Download citation * Received: 01 September 2017 * Revised: 05 December 2017 * Accepted:

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