Challenging interpretation of germline TP53 variants based on the experience of a national comprehensive cancer centre

TP53 variant interpretation is still challenging, especially in patients with attenuated Li–Fraumeni syndrome (LFS). We investigated the prevalence of pathogenic/likely pathogenic (P/LP) variants and LFS disease in the Hungarian population of cancer patients. By testing 893 patients with multiplex or familial cancer, we identified and functionally characterized novel splice variants of TP53 helping accurate variant classification. The differences among various semi-automated interpretation platforms without manual curation highlight the importance of focused interpretation as the automatic classification systems do not apply the TP53-specific criteria. The predicted frequency of the TP53 P/LP variants in Hungary is 0.3 per million which most likely underestimates the real prevalence. The higher detection rate of disease-causing variants in patients with attenuated LFS phenotype compared to the control population (OR 12.5; p < 0.0001) may raise the potential benefit of the TP53 genetic testing as part of the hereditary cancer panels of patients with multiple or familial cancer even when they do not meet Chompret criteria. Tumours developed at an earlier age in phenotypic LFS patients compared to the attenuated LFS patients which complicates genetic counselling as currently there are no different recommendations in surveillance protocols for LFS, phenotypic LFS, and attenuated LFS patients.


Challenging interpretation of germline TP53 variants based on the experience of a national comprehensive cancer centre
Henriett Butz 1,2,3,4,5* , Anikó Bozsik 1,3,5 , Vince Grolmusz 1,3 , Erika Szőcs 2 , János Papp 1,3 & Attila Patócs 1,3,4 TP53 variant interpretation is still challenging, especially in patients with attenuated Li-Fraumeni syndrome (LFS).We investigated the prevalence of pathogenic/likely pathogenic (P/LP) variants and LFS disease in the Hungarian population of cancer patients.By testing 893 patients with multiplex or familial cancer, we identified and functionally characterized novel splice variants of TP53 helping accurate variant classification.The differences among various semi-automated interpretation platforms without manual curation highlight the importance of focused interpretation as the automatic classification systems do not apply the TP53-specific criteria.The predicted frequency of the TP53 P/LP variants in Hungary is 0.3 per million which most likely underestimates the real prevalence.The higher detection rate of disease-causing variants in patients with attenuated LFS phenotype compared to the control population (OR 12.5; p < 0.0001) may raise the potential benefit of the TP53 genetic testing as part of the hereditary cancer panels of patients with multiple or familial cancer even when they do not meet Chompret criteria.Tumours developed at an earlier age in phenotypic LFS patients compared to the attenuated LFS patients which complicates genetic counselling as currently there are no different recommendations in surveillance protocols for LFS, phenotypic LFS, and attenuated LFS patients.
Classic Li-Fraumeni syndrome (LFS) is a high-penetrance hereditary condition predisposing to several tumour types in age-related phases 1 .In childhood, the characteristic tumour types are adrenocortical carcinoma, choroid plexus carcinoma, rhabdomyosarcoma, and medulloblastoma.During the transition from childhood to young adulthood, breast cancer, gastrointestinal, lung cancer, and different sarcomas are the most prevalent, while pancreatic and prostate cancer occur mostly during late adulthood 1 .The most characteristic tumour types are breast cancer, soft tissue sarcoma, osteosarcoma, brain tumour, and adrenocortical carcinoma which are called "core tumours".
So far, only germline TP53 pathogenic variants have been identified as disease-causing factors in association with this clinical condition 2 .As the clinical diagnosis is based on the presence of sarcoma in the proband and strong familial involvement (including at least two family members affected), the prevalence of the disease was estimated to be very rare without knowing the exact population frequency [3][4][5] .However, lately, pathogenic TP53 variants were identified in several patients with attenuated phenotypes due to the wide application of multigene panels used by next-generation sequencing in clinical practice even in patients not fulfilling the TP53 testing criteria 3 .Additionally, higher allele frequency of pathogenic/likely pathogenic (P/LP) TP53 variants was also described in population databases than it would have been expected based on the disease prevalence 4,5 .This fundamentally changed our view on disease penetrance; therefore, genetic testing criteria (Chompret criteria) were widened to the identification of cases irrespective of family history (Table 1) 2,3 .Recently, the use of the following phenotype categories was suggested by Kratz et al. 3 : (1) LFS: the presence of a germline P/LP or mosaic TP53 variant in a person with any cancer before age 18 years or who meets established testing criteria (Chompret A or B). ( 2) attenuated LFS: the presence of a germline P/LP or mosaic TP53 variant in a person with any cancer who does not meet LFS genetic testing criteria and has no cancer diagnosed before age 18 years.(3) incidental LFS: the presence of a germline P/LP or mosaic TP53 variant in a person/family without cancer.( 4) phenotypic LFS: the absence of a P/LP germline or germline mosaic TP53 variant in a person who meets classic LFS criteria or category A Chompret criteria.
For variant interpretation, several sources are used in daily routine including the National Institute of Health (NIH) funded endeavours, such as ClinVar (https:// www.ncbi.nlm.nih.gov/ clinv ar/) and Clinical Genome Resource (ClinGen, https:// clini calge nome.org/) 6 .ClinVar, is a freely accessible, public archive of reports on the relationships among human variations and phenotypes.It processes submissions reporting variants found in patient samples, assertions made regarding their clinical significance, information about the submitter, and other supporting data (https:// www.ncbi.nlm.nih.gov/ clinv ar/ intro/).It is probably the most widely used source for variant interpretation.However, due to its nature (individual submissions), discordant interpretation of variants has been often reported among ClinVar submitters and between expert reviewers [7][8][9][10] .Therefore, ClinGen performs curation of variant interpretation by variant curation expert panels, but the great majority of variants have been waiting for expert reviews.The American College of Medical Genetics and Genomics (ACMG) established guidelines for reporting and interpreting germline sequence variants in an effort to standardize clinical evaluation of genomic information which is recommended to use as a standard procedure 6,11 .Since then, different platforms (i.e.InterVar 12 , Varsome 13 and Franklin Genoox (https:// frank lin.genoox.com) have been created to apply ACMG criteria and to make variant interpretation easier.Due to the particular characteristics of the TP53 gene and the associated phenotypes (e.g., pleiotropic function and uncertain penetrance), specific criteria were recommended for variant classification 14,15 .Many P/LP variants and/or their interpretations are missing from the official NCI TP53 database or other databases.The functional consequences of these variants are also unknown.Certain gene and patient-specific factors have to be taken into consideration during variant classification, hence, variant interpretation is still challenging in routine molecular genetic diagnostics 16 , especially in case of patients not meeting Chompret criteria.
In the Hungarian population, the prevalence of the TP53 P/LP variants and the LFS disease are currently unknown.As a national genetic testing centre for cancer patients, we faced the abovementioned challenges.Therefore in this study our goals were (1) to assess the prevalence of TP53 P/LP variants in Hungarian patients with cancer; (2) to compare the TP53 detection rate in LFS, attenuated LFS, and phenotypic LFS population about which there is only scarce information, especially in a central European population; (3) to compare TP53 P/LP rate in our cohort to non-cancer and disease-control groups; (4) to perform RNA-level characterization of novel TP53 splice variants to aid the correct variant classification; and (5) to investigate the clinical importance of applying TP53-specific interpretation compared to semi-automatic web-based platforms routinely used in diagnostic genetic testing laboratories.

TP53 variant characterization and variant interpretation. Germline genotyping of the TP53
gene of 893 patients with multiplex or familial cancer resulted in 15 (P/LP/VUS) variants in 16 patients (NM_000546.6:c.743G> A was observed in two patients) (Table 2).Of the 15 variants, only 6 variants were present in the NCI TP53 database 17 (Table 3).
The variant classification was performed including these functional data as well following the specifications of the ACMG/AMP variant interpretation guidelines for germline TP53 variants.All variants were submitted to the LOVD database.
TP53 variant detection rate and differences in interpretation in patients with Li-Fraumeni spectrum.Of the 893 patients, 13 met Chompret A criteria.Among them, TP53 P/LP variants were Table 1.Chompret criteria.In our study, we investigated patients fulfilling category A Chompret criteria resembling the "classic" LFS phenotype.
Category A Proband with a core tumour before age 46 years and ≥ 1 first-or second-degree relative with a core tumour (except a breast cancer if proband had breast cancer) before age 56 years OR ≥ 2 tumours (not multiple breast cancers), including 2 core tumours, the first of which occurred before age 46 years Category B ANY of the following: Adrenocortical carcinoma, choroid plexus carcinoma, anaplastic rhabdomyosarcoma, breast cancer before age 31 years, osteosarcoma, childhood hypodiploid acute lymphoblastic leukaemia, sonic hedgehog-medulloblastoma  2a).None of these patients met classic LFS criteria.Among 880 patients who did not meet Chompret A criteria (Table 4), TP53 variants were identified in 9 cases: 5 P/LP (5/880, 0.5%) and 4 VUS.The difference in detection rate between patients meeting and not meeting Chompret A criteria indicates the good applicability for the indication of the genetic test (p < 0.0001).Two control groups were investigated to compare this data to the allele frequencies.As population control data, the Genome Aggregation Database (gnomAD V.2.1.1)was used applying the European non-Finnish non-cancer population (n = 59,095) 18 .As a disease control group clinical data of Li-Fraumeni patients carrying germline TP53 variants were obtained from the US National Cancer Institute (NCI) TP53 Database (a successor of the International Agency for Research on Cancer TP53 database) 17 .This database contained data from 4455 patients with germline TP53 variants, of these 213 patients carried the variants we found in our investigated population.In population  controls, TP53 P/LP variants were identified in 27 of the 59,095 cases (0.05%) (Supplementary Table S1), therefore the odds to detect TP53 P/LP variant in cancer patients with multiple tumours or patients having multiplex tumours in the family but not fulfilling Chormpet A criteria was 12.5 (OR 12.5; 95% CI 4.803-32.54;p < 0.0001).By comparing literature resources regarding TP53 variant interpretation we found remarkable differences according to the often-used web resources.Namely, 13% (2/15), 20% (3/15), and 27% (4/15) of the variants referred by Varsome, Franklin, and ClinVar respectively, were misclassified (VUS instead of P/LP or vice versa) without manual curation compared to the gene-specific interpretation results (Table 3).

Genotype-phenotype correlations. LFS and attenuated phenotypes were not associated with variants'
localization in the TP53 gene, as the identified genetic alterations were located throughout the whole gene irrespective of the phenotype category (Fig. 2b).
However, the age of the first tumour onset was younger in patients with LFS compared to ones having an attenuated form of the disease.The probability of tumour development and the average age of onset of the first tumour in phenotypic LFS patients (TP53 wild-type genotype) was between LFS and attenuated LFS patients (harbouring TP53 P/LP variant) (Fig. 2c,d).We did not find any association between the age of the first tumour onset and different TP53 variants either among our cases or cases registered in the NCI TP53 database (Fig. 2e).Phenotypes were not related to the number of tumours either in the probands or in the family of the probands (Fig. 2f).The lack of genotype-phenotype associations is additionally strengthened by the identification of the same pathogenic variant (NM_000546.6:c.743G> A) in a LFS and an attenuated LFS patient.
Among individuals fulfilling Chompret A criteria, there were no differences in multiple tumours occurrence, familial appearance, rare tumour type, or early-onset breast tumour development between LFS (TP53 P/LP carriers) and phenotypic LFS patients (TP53 wild-type genotype).We found the same relations when all TP53 P/LP carriers (LFS together attenuated LFS group) were compared to TP53 wild-type individuals (phenotypic LFS group).

Discussion
Altogether we identified 12 different P/LP variants in our cancer patient cohort.In two cases functional in vitro characterization resulted in downgrading of classification category.Indeed, despite the strong effect indicated by splice predictors for variants NM_000546.6:c.375 + 6T > C and NM_000546.6:c.376-2dupA, this could not be corroborated by in vitro functional assays, indicating that in vitro testing of the splice effect is an important part of accurate variant interpretation.
Based on literature data, the P/LP TP53 variant detection rate was reported as 17% (82/474 19 ), 21% (22/105 20 ), 29% (67/232 21 ), or 35% (69/195 22 ) in patients fulfilling Chompret criteria.In our patient cohort, this was higher (54%) which could be explained by the patient selection criteria as in our study only individuals fulfilling the Chompret A category were included (patients fulfilling Chomrpet B criteria were omitted due to the low detection rate).The prevalence of LFS in Hungary is unknown.In 4 years, we identified 13 probands fulfilling the Chompret A criteria.Our center as a national reference center in Hungary performs germline TP53 testing.Therefore, the 13 cases of LFS syndrome identified between 2018 and 2022 in Hungary (having approx.10 million inhabitants) estimate the incidence as 0.32 per million.This data most likely underestimates the real prevalence, however, the 5 cases out of the 880 unselected cancer patients with P/LP TP53 variants identified during 1 year would mean 0.5% prevalence.Further epidemiological studies are warranted to determine the real prevalence of LFS in Hungary.Our P/LP TP53 variant frequency data observed in cancer patients are in line with earlier published data.The 0.5% incidence rate detected using multigene panel testing strategy in our consecutive cancer patients is similar to those reported by Rana et al.They detected 126 TP53 mutant cases out of 40,885 tested cancer patients (0.3%) 23 .www.nature.com/scientificreports/ We found a higher P/LP TP53 variant detection rate in cancer patients not fulfilling Chompret criteria (attenuated LFS phenotype) compared to the non-cancer GnomAD control population (OR 12.5; p < 0.0001).This raises the potential benefit of the TP53 genetic testing as part of the hereditary cancer panel of patients with multiple or familial cancer even when they do not meet Chompret criteria.Identifying most of the cases with the germline P/LP TP53 variant allows in these families, the possibility for genetic counselling followed by genetic testing for asymptomatic first-degree relatives.Then, in mutation carriers the most adequate clinical interventions can be started, hence the best prevention can be achieved.
The identified differences among different semi-automated interpretation platforms with and without manual curation highlight the importance of focused interpretation especially, since the automatic classification systems apply "classic" ACMG classification criteria 11 instead of TP53-specific ACMG criteria 14 .In addition, patient and family-specific data together with in vitro functional data should be included for precise variant interpretation [14][15][16] .
In our cohort, we did not observe genotype-phenotype correlation except that, expectedly, the first tumour onset was earlier in LFS patients compared to attenuated LFS individuals.Interestingly, tumours developed at an earlier age in phenotypic LFS patients (TP53 wild-type genotype) compared to attenuated LFS patients (harbouring TP53 P/LP variant).Fortuno et al. observed similar findings, patients carrying truncating and hotspot variants experienced an earlier cancer diagnosis and might be more prone to present with LFS malignancies compared to carriers of other TP53 variant types 24 .However, the authors concluded that the observed differences were minor, and current evidence was insufficient to consider genotype-phenotype associations to assist with the clinical management of TP53 carriers 24 .These observations complicate genetic counselling as currently there are no distinguished recommendations in surveillance protocols for LFS, phenotypic LFS, and attenuated LFS patients 2,3,18 .However, our findings raised the possibility of delayed starting age for surveillance protocols in attenuated LFS cases that would relieve the burden of the exhausting childhood surveillance program in those patients and their families.Naturally, this should be further validated on larger cohorts, consensual international findings, and longer follow-up of cancer patients with attenuated LFS phenotype.
Study limitations should be declared, namely, the small sample number limits the assessment of genotype-phenotype associations.Our centre is the only Comprehensive Cancer Centre in Hungary, performing germline testing for cancer patients and it is assumed that all cases with a suspicion of having LFS might be referred to our centre, but we feel that not all cases have been referred during the tested period.Further efforts (including further development of the nationwide cancer registry and education of members of regional cancer centres) are needed for better estimates of the real prevalence of rare cancer types in Hungary.Despite that all results with P/LP variants and VUSs were validated on a second independently extracted DNA sample using Sanger sequencing (showing 100% concordant results with the NGS method), we cannot exclude entirely the possibilities of age-and/or therapy-related clonal haematopoiesis.

Materials and methods
Subjects.TP53 gene was investigated in 880 consecutive oncology patients referred for molecular genetic testing at our national centres (Department of Laboratory Medicine, Semmelweis University and Department of Molecular Genetics, National Institute of Oncology) between 2021 and 2022.This cohort consisted of patients with potential hereditary tumour predisposition.Their genetic analysis was performed by a multigene panel within routine clinical genetic care.
Targeted TP53 analysis was performed in 13 patients with multiple tumours or multiplex tumours in the family where clinicians suspected Li-Fraumeni syndrome (fulfilling Chompret A criteria) between 2018 and 2022 (Table 2) according to Kratz et al. 3 .In our study, we investigated patients fulfilling category A criteria resembling the "classic" LFS phenotype and patients fulfilling Chomrpet B criteria were omitted due to the low detection rate.The phenotypic classification was applied following the recommendation by Kratz et al. 3 .
According to Hungarian legal and ethical regulations, germline genetic analysis was performed following genetic counselling.Each patient gave informed consent to the genetic test based on the approval of the Scientific and Research Committee of the Medical Research Council of the Ministry of Health, Hungary (ETT-TUKEB 53720-4/2019/EÜIG, ETT-TUKEB 4457/2012/EKU).
All research was performed in accordance with the Declaration of Helsinki and informed consent was obtained from all participants.
To compare allele frequencies, two control groups were investigated.Population control data from Genome Aggregation Database (gnomAD V.2.1.1)was used applying the European non-Finnish non-cancer population (n = 59,095) 18 .As a disease control group clinical data of Li-Fraumeni patients carrying germline TP53 variants were obtained from the US National Cancer Institute (NCI) TP53 Database (a successor of the International Agency for Research on Cancer TP53 database) 17 containing data from 4455 patients with germline TP53 variants.
Data analysis was run on Illumina BaseSpace Dragen Germline pipeline v2.1, and variants were called between 20 and 70% variant allele frequency (VAF), and validated by conventional Sanger sequencing.All investigated VAF ratios were close to 50% except in one case, where VAF was 22%.In this case, unfortunately,

Figure 1 .
Figure 1.Validation of the germline TP53 variants c.275 + 6T > C and c.376-2dupA by Sanger sequencing (a) and RNA level characterizations of TP53 splice variants (b) Agarose gel electrophoresis of the RT-PCR products of the TP53:c.375+ 6T > C variant carrier and controls.A very faint, 200 bp-shorter extra band is detectable at the c.375 + 6T > C variant carrier sample (c) Sanger sequencing of the RT-PCR product of the TP53:c.375+ 6T > C variant carrier.The extremely low level of aberrant splice product of the variant carrier, generated by weakening the canonical splice donor site and applying a pre-existing cryptic exonic donor GT site (shown with red bracket, chromosome position is indicated) (d) Screenshot of the TP53 exon 4 genomic region (hg 19) visualized by UCSC genome browser (https:// genome-euro.ucsc.edu).The orange arrow indicates the position of the activated donor GT splice site, purple arrow marks the variant position.The measure of the aberrant splicing, although detectable, is extremely low, therefore negligible from a clinical point of view.

Figure 2 .
Figure 2. (a) Patient cohort and genetic testing outcome.(b) Localizations of the identified variants in the TP53 gene.(c,d) Age and probability of first tumour onset in LFS, phenotypic LFS, and attenuated LFS groups.(e) Age of first tumour onset according to variants found in NCI TP53 database.Green stars-attenuated LFS; red stars-LFS patients.(f) Number of tumours in the proband and probands' families in LFS, phenotypic and attenuated LFS groups.

Table 2 .
Patient characteristics.CNS central nervous system, LFS Li-Fraumeni syndrome, A Chompret A category, M male, F female.

Table 3 .
Variant characteristics.VCEP Variant Curator Expert Panel, MAF minor allele frequency, P/LP pathogenic/likely pathogenic, VUS variant of uncertain significance, DNE dominant negative effect, LOF lossof-function.Bold letters indicate downgrading following manual curation of interpretation using gene-specific variant interpretation criteria.In the 'TP53-specific interpretation' column ACMG classification criteria and evidence levels are indicated in parenthesis: str strong, supp supporting, mod moderate.

Table 4 .
Characteristics of patients not fulfilling Chompret A criteria.HBOC hereditary breast and ovarian cancer syndrome, n number, y years.