Genotypic and Phenotypic Characteristics of Germline TP53 Variant Carriers: Experience from Two Cancer Genetic Counseling Units

Genotypic and Phenotypic Characteristics of Germline TP53 Variant Carriers: Experience from Two Cancer Genetic Counseling Units | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Genotypic and Phenotypic Characteristics of Germline TP53 Variant Carriers: Experience from Two Cancer Genetic Counseling Units Beatriz Grau Mirete, Asia Ferrández Arias, Paula Rodríguez Payá, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7781365/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Germline TP53 pathogenic or likely pathogenic variants underlie Li–Fraumeni syndrome (LFS), but with broader use of multigene panel (MGP) testing carriers are increasingly identified outside classic criteria. We retrospectively studied 36 carriers from 18 families evaluated at two Spanish hereditary cancer units (2005–2023). Clinical data were obtained from medical records, and variants were functionally classified using the Giacomelli model and IARC thresholds as dominant-negative with loss of function (DNE_LOF), non-dominant-negative without loss of function (noDNE_noLOF), non-dominant-negative with loss of function, or not assessed. Descriptive statistics summarized the tumor spectrum and group comparisons were performed with the Mann–Whitney U test (α=0.05). Overall, 23 of 36 carriers (63.9%) developed at least one malignancy; 58.3% were female and the median age at first cancer was 36 years (range 27–54). Patients identified through MGP testing had later onset compared with those meeting clinical LFS criteria (median 42 [37–58] vs 27 [4–30] years; p=0.011). Breast cancer was the most frequent tumor (65% of first cancers, median age 37 [30–47]), whereas none of the MGP-ascertained carriers developed LFS “core” tumors (sarcoma, brain tumor, adrenocortical carcinoma). Fifteen distinct germline variants were identified; 80% were missense, mainly in exons 5–8. DNE_LOF (median 30 [25–38.5]) and not-assessed variants (32 [27–48]) were associated with earlier onset compared with noDNE_noLOF (47 [37–63]; p=0.034). Eight carriers (34.7%) developed second primaries after a median interval of 6 years, and four developed third primaries. Functional classification stratifies risk, supporting a broader TP53-related hereditary cancer syndrome and individualized surveillance. TP53 Li–Fraumeni Germline variants Hereditary cancer Genotype–phenotype correlation Multigene panel testing Figures Figure 1 Figure 2 Figure 3 Introduction Li–Fraumeni syndrome (LFS) is an autosomal dominant cancer‑predisposition syndrome caused by germline pathogenic/likely pathogenic variants (P/LPVs) in TP53 , and is characterized by early‑onset “core” tumors—sarcomas, premenopausal breast cancer ≤31 years, adrenocortical carcinoma, and brain tumors.[1,2] Historically, ascertainment relied on clinical criteria (classic LFS; revised Chompret), which enriched for pediatric cancers and very early onset disease.[2] In contemporary practice, the widespread use of multigene panel (MGP) testing is uncovering TP53 carriers who do not meet traditional criteria, revealing a broader phenotypic spectrum with later ages at diagnosis and fewer core tumors than expected under classic LFS.[3,4,5] Beyond clinical heterogeneity, TP53 variants are functionally diverse. The p53 protein is a central tumor suppressor that coordinates cell‑cycle arrest, apoptosis, senescence, and DNA damage responses.[6] Experimental models integrating dominant‑negative effect (DNE) and loss‑of‑function (LOF) have enabled functional grouping of germline variants (e.g., DNE_LOF, noDNE_LOF, noDNE_noLOF).[7] These groupings have been incorporated into curation frameworks and reference resources (ClinGen TP53 VCEP; IARC TP53 database) to improve risk interpretation.[8,9] Notably, some population variants—such as the Brazilian founder p.R337H—fall into non‑DNE classes and are linked to distinctive, often later‑onset patterns and tumor spectra.[10] Against this backdrop, real‑world data are needed to clarify how ascertainment pathways and functional class relate to clinical expression. We report the tumor spectrum and age at first cancer in TP53 P/LPV carriers managed across two Spanish hereditary cancer units, comparing MGP‑ascertained carriers with those fulfilling clinical criteria, and testing whether DNE_LOF variants associate with earlier onset and core‑tumor enrichment relative to non‑DNE classes. Our goal is to provide concise, clinically actionable evidence to support risk stratification and individualized surveillance in TP53 ‑related hereditary cancer. Methods We performed a retrospective cohort study including 36 carriers of germline TP53 P/LPVs from 18 unrelated families who attended two Spanish hereditary cancer units (Hospital General Universitario de Elche and Hospital Clínico Universitario de Valencia) between 2005 and 2023. Clinical data (demographics, personal and family cancer history, tumor dates and types, treatments, outcomes) were extracted from electronic medical records using a standardized template. The primary endpoint was age at first malignant neoplasm; secondary endpoints were tumor spectrum (first and subsequent primaries), frequency of “core” LFS tumors, intervals between successive primaries, and distributions by ascertainment pathway and functional class. The research question was whether age at onset and tumor spectrum differ (i) by ascertainment pathway and (ii) by functional class of the TP53 variant. We hypothesized that carriers identified through multigene panel (MGP) testing would present later and with fewer core tumors than those fulfilling clinical criteria, and that dominant-negative, loss-of-function (DNE_LOF) variants would be associated with earlier onset and enrichment for core tumors compared with non-dominant-negative, non-loss-of-function (noDNE_noLOF) variants. Inclusion required a documented germline TP53 P/LPV classified as pathogenic/likely pathogenic in ClinVar, either by one or more accredited clinical genetic testing laboratories or by the ClinGen TP53 Variant Curation Expert Panel (VCEP); variants of uncertain significance were excluded.[9,11] Carriers were grouped by ascertainment as: MGP testing in oncology; cascade testing for a known familial variant; clinical LFS (classic or 2015 revised Chompret criteria); or tumor-first detection with subsequent germline confirmation.[1,2] Core LFS tumors were defined as sarcoma, premenopausal breast cancer, adrenocortical carcinoma (ACC), and primary brain tumors.[1] Unique variants were annotated by type and exon location and mapped to functional categories using the Giacomelli experimental model (integration of dominant-negative effect and loss-of-function) harmonized with IARC thresholds: DNE_LOF, noDNE_LOF, noDNE_noLOF; variants not evaluated in the assay were labeled “not assessed” (NA). The Brazilian founder variant p.R337H (c.1010G>A) was categorized as noDNE_noLOF.[7,8,9] Continuous variables were summarized as medians (ranges or IQRs) and compared using two-sided Mann–Whitney U tests (α=0.05); categorical variables were compared with Fisher’s exact tests. For >2-group comparisons across functional classes, pairwise Mann–Whitney tests with Holm–Bonferroni correction were prespecified; where cell sizes were sparse, findings were summarized descriptively. Analyses were performed using IBM SPSS Statistics v28. This study was approved by the ethics committees of Hospital General Universitario de Elche and conducted in accordance with the principles of the Declaration of Helsinki. Given the exclusive use of de-identified data collected during routine care, a waiver of informed consent was granted. Results Among the 36 carriers analyzed, women represented 58.3% and men 41.7%. Indications for genetic testing were cascade testing in 20 individuals (55.6%), MGP testing in 8 (22.2%), fulfillment of clinical LFS criteria in 7 (19.4%), and tumor-first detection with subsequent germline confirmation in 1 (2.8%). Of the MGP-ascertained carriers, seven were women with breast cancer and one was tested due to colonic polyposis. Overall, 23/36 (63.9%) developed at least one malignant neoplasm (17 women, 6 men). The median age at first cancer was 36 years (range 27–54); by sex, 37 (30–52.5) for women and 30.5 (4–54) for men. By ascertainment, median age at first cancer was 42 (37–58) for MGP, 41 (33–60.5) for cascade, and 27 (4–30) for clinical LFS; the difference between MGP and clinical LFS was significant ( p = 0.011). First-tumor spectrum is summarized in Figure (Fig.) 1 : breast cancer predominated (15/23, 65.2%; median 37 [30–47] years), with two ACC, two brain tumors, and single cases of pancreatic, gastric, colorectal, and bladder cancers. Eight of the 23 affected carriers (34.7%) developed a second primary (7 women, 1 man), and four women (17.3%) developed a third primary ( Fig. 1 ). Among the eight with second primaries, 50% (4/8) were identified through MGP testing, 37.5% (3/8) met clinical criteria, and 12.5% (1/8) were tested due to a known familial variant. Breast cancer was the most common second primary (5/8, 62.5%); none had undergone prophylactic mastectomy prior to the second event. One second primary was a brain tumor and one a sarcoma (both LFS “core” tumors). No MGP-ascertained carrier developed a core tumor. The median interval between first and second primaries was 6 years (range 3–9), and between second and third primaries 5.5 years (2–9). We identified 15 distinct germline TP53 P/LPVs; 12/15 (80%) were missense, with ~60% mapping to exons 5–8. The Brazilian founder variant p.R337H (c.1010G>A) was present in four carriers from two families. The per-variant catalog with exon position, type, Giacomelli functional class, and number of carriers is shown in Fig. 2 , and class-level summaries are provided in Fig. 3 . Functional grouping comprised DNE_LOF in 6/15 (40%), noDNE_noLOF in 4/15 (26.6%), noDNE_LOF in 1/15 (6.6%), and not assessed (NA) in 4/15 (26.6%). Genotype–phenotype analyses ( Fig. 3 ) indicated earlier ages at first cancer for DNE_LOF (median 30 years [25–38.5]) and NA (32 [27–48]) compared with noDNE_noLOF (47 [37–63]); the DNE_LOF versus noDNE_noLOF contrast was significant ( p = 0.034). Among women with breast cancer, earlier ages were observed for DNE_LOF (median 34 [28.5–49.5]) and NA (31 [27–37]) compared with noDNE_noLOF (46.5 [37–63]). Core tumors were enriched among DNE_LOF carriers, with exceptions of one sarcoma in NA and one ACC in a p.R337H carrier. Among those with second primaries, 4/8 (50%) were DNE_LOF, 2/8 (25%) noDNE_noLOF, and 2/8 (25%) NA; third primaries occurred in 2/4 DNE_LOF and 2/4 NA carriers. Within the MGP subset, 50% of variants were noDNE_noLOF, consistent with the older age at onset and milder spectrum in that group. At last follow-up, 8/23 (34.7%) affected carriers had died (median age at death 56 years [42–62]); two deaths were non-cancer (stroke). Discussion Our study adds evidence that clinical expression among germline TP53 carriers varies with both variant function and ascertainment. In our series, individuals identified by MGP testing presented later and did not develop core LFS tumours, a pattern consistent with panel‑based cohorts in which carriers are older at diagnosis and less likely to satisfy classic criteria [3]. Conversely, carriers from clinically ascertained LFS families showed earlier onset and more core tumours, echoing previous observations [12,13]. Taken together, these findings support a spectrum of heritable TP53 ‑related cancer risk in which genotype and ascertainment inform comprehensive surveillance throughout adulthood. Differences in variant function account for much of the heterogeneity seen in germline TP53 . This pattern is consistent with foundational functional maps. Kato et al. performed a high‑throughput yeast transactivation assay covering thousands of single‑amino‑acid substitutions and showed that many DNA‑binding domain missense variants have severe loss of transcriptional activity and dominant‑negative behavior [14]. Kotler et al. used deep mutational scanning in mammalian cells to survey near‑complete missense space and linked measured functional impact to human tumor hotspot frequencies [15]. Other works further show that mutant p53 can drive genomic instability and acquire oncogenic gain‑of‑function properties beyond simple haploinsufficiency [16,17]. At the clinical level, a large observational cohort reported that LOF (with or without DNE) variants were associated with significantly earlier age at first cancer than variants retaining transactivation, reinforcing a functional gradient of penetrance [5]. Complementing this, a retrospective, panel‑based series found that panel‑ascertained TP53 carriers were older at first diagnosis and less likely to meet classic LFS criteria than clinically ascertained families [3]. Our data mirror these trends, none of the MGP‑ascertained carriers developed core tumors, and their first cancers occurred later, while DNE_LOF versus noDNE_noLOF showed a 17‑year median age difference ( p =0.034). Variants without assay data in our study behaved more like DNE_LOF than noDNE_noLOF—similar to other disruptive alleles in large cohorts—but expert reviews caution that functional class should guide, not determine, management until prospective, variant-stratified data are available [18]. The Brazilian founder p.R337H illustrates how a single allele can shape population‑level risk. Achatz et al. first characterized family‑based series from Southern/Southeastern Brazil showing that p .R337H segregates with LFS and LFS-like phenotypes yet often with later adult onset than canonical DNA‑binding mutations [19]. A landmark statewide neonatal screening and surveillance program demonstrated that identifying p .R337H carriers at birth facilitated early‑stage detection of pediatric ACC and improved outcomes compared with historical controls [20]. Broader institutional cohorts confirm that p .R337H families manifest a bimodal pattern—substantial risk for childhood ACC plus adult cancers (e.g., breast, brain, sarcoma) that tend to arise later than in classic LFS, with some series noting higher frequencies of thyroid or renal cancers [10,21]. Our experience was concordant: two of four p .R337H carriers developed malignancies (ACC at 1 year; breast cancer at 36 years), showing the need for variant‑aware counseling (e.g., pediatric adrenal screening) layered onto core LFS surveillance. Consensus statements recommend annual whole‑body MRI (WBMRI), periodic brain MRI, targeted organ imaging, and age‑appropriate biochemical screening from childhood onward, with adaptation to local resources [22,23]. Evidence for benefit comes from prospective observational studies by Villani et al.: the initial report and 11‑year update showed that structured surveillance detects more asymptomatic, early‑stage tumors and is associated with markedly higher 5‑year overall survival (~89% under surveillance vs ~60% in those declining) compared with historical/unscreened comparators [12,13]. Therapeutic decisions also benefit from a TP53 ‑specific lens. A consensus review (Thariat et al.) and a multi‑institution retrospective series (Hendrickson et al.) suggest that when clinically equivalent alternatives exist, radiotherapy (RT) should be avoided or adapted to reduce radiation‑associated second primaries; if RT is necessary, dose/fields should be minimized with vigilant follow‑up [24,25]. For breast cancer—the most common first malignancy in our study—a large analysis reported that TP53 ‑associated tumors are often high‑grade and HER2‑positive, supporting early MRI‑based screening and, in selected carriers, discussion of bilateral risk‑reducing mastectomy to avoid adjuvant RT and lower contralateral risk [26]. Alongside these genotype–phenotype observations, two interpretive caveats remain central when applying TP53 testing in clinical settings. Low-allele-fraction TP53 calls in blood can reflect clonal hematopoiesis or post-zygotic mosaicism rather than true germline, and several clinical series have shown that orthogonal testing in non-hematopoietic tissue (such as fibroblasts or saliva) is essential to avoid misclassifying somatic events as heritable LFS [27,28]. The main limitations of this study include its retrospective design, small sample size, and potential referral bias, which limit statistical power and generalizability. Reliance on electronic medical records may have introduced variability in the level of clinical detail, and germline status was inferred from blood only without orthogonal confirmation, while some variants could not be functionally classified. Despite these limitations, the study has strengths: it draws on two hereditary cancer units, combines carriers identified through both clinical criteria and multigene panel testing, and applies functional classification to explore genotype–phenotype correlations. These features provide useful real-world insights and support the need for larger, prospective, standardized studies to validate and extend our findings. Conclusion This study shows that clinical expression among germline TP53 P/LPV carriers is influenced by both functional class and ascertainment pathway. DNE_LOF and NA variants were associated with earlier onset and a higher frequency of core LFS tumors, whereas carriers identified through MGP testing, often with noDNE_noLOF variants, presented later and with milder phenotypes. These findings emphasize the heterogeneity of TP53 -related hereditary cancer and support incorporating functional classification into risk assessment to better tailor surveillance and counseling strategies. Declarations Submitting Declarations: We confirm that the manuscript is original, has not been published previously, and is not under consideration elsewhere. All authors have read and approved the final version of the manuscript, and declare that they have no conflicts of interest. Funding: The authors received no financial support for the research, authorship, and/or publication of this article. References Sánchez-Heras AB, Ramon Y Cajal T, Pineda M, et al. SEOM clinical guideline on heritable TP53-related cancer syndrome (2022). Clin Transl Oncol . 2023;25(9):2627-2633. doi:10.1007/s12094-023-03202-9 Bougeard G, Renaux-Petel M, Flaman JM, et al. Revisiting Li-Fraumeni Syndrome From TP53 Mutation Carriers. J Clin Oncol . 2015;33(21):2345-2352. doi:10.1200/JCO.2014.59.5728 Rana HQ, Gelman R, LaDuca H, et al. Differences in TP53 Mutation Carrier Phenotypes Emerge From Panel-Based Testing. J Natl Cancer Inst . 2018;110(8):863-870. doi:10.1093/jnci/djy001 Rana HQ, Clifford J, Hoang L, et al. Genotype-phenotype associations among panel-based TP53+ subjects. Genet Med . 2019;21(11):2478-2484. doi:10.1038/s41436-019-0541-y de Andrade KC, Khincha PP, Hatton JN, et al. Cancer incidence, patterns, and genotype-phenotype associations in individuals with pathogenic or likely pathogenic germline TP53 variants: an observational cohort study. Lancet Oncol . 2021;22(12):1787-1798. doi:10.1016/S1470-2045(21)00580-5 Olivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. Cold Spring Harb Perspect Biol . 2010;2(1):a001008. doi:10.1101/cshperspect.a001008 Giacomelli AO, Yang X, Lintner RE, et al. Mutational processes shape the landscape of TP53 mutations in human cancer. Nat Genet . 2018;50(10):1381-1387. doi:10.1038/s41588-018-0204-y Bouaoun L, Sonkin D, Ardin M, et al. TP53 Variations in Human Cancers: New Lessons from the IARC TP53 Database and Genomics Data. Hum Mutat . 2016;37(9):865-876. doi:10.1002/humu.23035 Fortuno C, Lee K, Olivier M, et al. Specifications of the ACMG/AMP variant interpretation guidelines for germline TP53 variants. Hum Mutat . 2021;42(3):223-236. doi:10.1002/humu.24152 Ferreira AM, Brondani VB, Helena VP, et al. Clinical spectrum of Li-Fraumeni syndrome/Li-Fraumeni-like syndrome in Brazilian individuals with the TP53 p.R337H mutation. J Steroid Biochem Mol Biol . 2019;190:250-255. doi:10.1016/j.jsbmb.2019.04.011 Landrum MJ, Lee JM, Benson M, et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res . 2018;46(D1):D1062-D1067. doi:10.1093/nar/gkx1153 Villani A, Tabori U, Schiffman J, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: a prospective observational study. Lancet Oncol . 2011;12(6):559-567. doi:10.1016/S1470-2045(11)70119-X Villani A, Shore A, Wasserman JD, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: 11 year follow-up of a prospective observational study. Lancet Oncol . 2016;17(9):1295-1305. doi:10.1016/S1470-2045(16)30249-2 Kato S, Han SY, Liu W, et al. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. Proc Natl Acad Sci U S A . 2003;100(14):8424-8429. doi:10.1073/pnas.1431692100 Kotler E, Shani O, Goldfeld G, et al. A Systematic p53 Mutation Library Links Differential Functional Impact to Cancer Mutation Pattern and Evolutionary Conservation. Mol Cell . 2018;71(1):178-190.e8. doi:10.1016/j.molcel.2018.06.012 Zerdoumi Y, Lanos R, Raad S, et al. Germline TP53 mutations result into a constitutive defect of p53 DNA binding and transcriptional response to DNA damage. Hum Mol Genet . 2017;26(14):2591-2602. doi:10.1093/hmg/ddx106 Muller PA, Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities. Cancer Cell . 2014;25(3):304-317. doi:10.1016/j.ccr.2014.01.021 Fortuno C, Pesaran T, Mester J, et al. Genotype-phenotype correlations among TP53 carriers: Literature review and analysis of probands undergoing multi-gene panel testing and single-gene testing. Cancer Genet . 2020;248-249:11-17. doi:10.1016/j.cancergen.2020.09.002 Achatz MI, Olivier M, Le Calvez F, et al. The TP53 mutation, R337H, is associated with Li-Fraumeni and Li-Fraumeni-like syndromes in Brazilian families. Cancer Lett . 2007;245(1-2):96-102. doi:10.1016/j.canlet.2005.12.039 Custódio G, Parise GA, Kiesel Filho N, et al. Impact of neonatal screening and surveillance for the TP53 R337H mutation on early detection of childhood adrenocortical tumors. J Clin Oncol . 2013;31(20):2619-2626. doi:10.1200/JCO.2012.46.3711 Formiga MNDC, de Andrade KC, Kowalski LP, Achatz MI. Frequency of Thyroid Carcinoma in Brazilian TP53 p.R337H Carriers With Li Fraumeni Syndrome. JAMA Oncol. 2017;3(10):1400-1402. doi:10.1001/jamaoncol.2016.6389 Kratz CP, Achatz MI, Brugières L, et al. Cancer Screening Recommendations for Individuals with Li-Fraumeni Syndrome. Clin Cancer Res . 2017;23(11):e38-e45. doi:10.1158/1078-0432.CCR-17-0408 Frebourg T, Bajalica Lagercrantz S, Oliveira C, Magenheim R, Evans DG; European Reference Network GENTURIS. Guidelines for the Li-Fraumeni and heritable TP53-related cancer syndromes. Eur J Hum Genet. 2020;28(10):1379-1386. doi:10.1038/s41431-020-0638-4 Thariat J, Chevalier F, Orbach D, et al. Avoidance or adaptation of radiotherapy in patients with cancer with Li-Fraumeni and heritable TP53-related cancer syndromes. Lancet Oncol. 2021;22(12):e562-e574. doi:10.1016/S1470-2045(21)00425-3 Hendrickson PG, Luo Y, Kohlmann W, et al. Radiation therapy and secondary malignancy in Li-Fraumeni syndrome: A hereditary cancer registry study. Cancer Med. 2020;9(21):7954-7963. doi:10.1002/cam4.3427 Breast Cancer Association Consortium, Mavaddat N, Dorling L, et al. Pathology of Tumors Associated With Pathogenic Germline Variants in 9 Breast Cancer Susceptibility Genes. JAMA Oncol . 2022;8(3):e216744. doi:10.1001/jamaoncol.2021.6744 Weitzel JN, Chao EC, Nehoray B, et al. Somatic TP53 variants frequently confound germ-line testing results. Genet Med. 2018;20(8):809-816. doi:10.1038/gim.2017.196 Mester JL, Jackson SA, Postula K, et al. Apparently Heterozygous TP53 Pathogenic Variants May Be Blood Limited in Patients Undergoing Hereditary Cancer Panel Testing. J Mol Diagn. 2020;22(3):396-404. doi:10.1016/j.jmoldx.2019.12.003 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 26 Oct, 2025 Reviewers agreed at journal 09 Oct, 2025 Reviewers invited by journal 09 Oct, 2025 Editor assigned by journal 08 Oct, 2025 Submission checks completed at journal 08 Oct, 2025 First submitted to journal 04 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7781365","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":532783529,"identity":"7af76bd3-8028-4ae5-9e3d-c5d2c3bbb2ce","order_by":0,"name":"Beatriz Grau Mirete","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYBACAygtx8/eAOJaEK/FWLLnAIgrQbyWxA0zEkA0EVrM2Zsfv66oOZy4QfL51Q0/CiQY+Nu7E/Bqsew5ZmZ55thh4+3SOWU3e4AOkzhzdgN+h91IMDNsYDssu3N2TtoNHqAWA4lcAlruP/9m2PDvMOOGm2fSbv4hSssNHuOHjW2HFTfcYD92myhbLHtyyhgb+9KBgZzDdlvGQIKHoF/M2Y9v/tjwzRoYlcef3Xzzx0aOv70XvxYgYIPGBQ84jngIKQcB5g8Qmv0BMapHwSgYBaNgBAIArUhM0L0ggggAAAAASUVORK5CYII=","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":true,"prefix":"","firstName":"Beatriz","middleName":"Grau","lastName":"Mirete","suffix":""},{"id":532783530,"identity":"01d4727e-ea9a-4b4b-9a9b-b37db5a8b9c9","order_by":1,"name":"Asia Ferrández Arias","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"Asia","middleName":"Ferrández","lastName":"Arias","suffix":""},{"id":532783531,"identity":"a86479e6-1ceb-4639-adf0-d11c58106d97","order_by":2,"name":"Paula Rodríguez Payá","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"Paula","middleName":"Rodríguez","lastName":"Payá","suffix":""},{"id":532783532,"identity":"083d6d82-2c24-4e32-92ac-0820abd206a2","order_by":3,"name":"María Valero Revert","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Valero","lastName":"Revert","suffix":""},{"id":532783533,"identity":"2fd6813c-063a-4121-8f19-13273874afee","order_by":4,"name":"David Sánchez Garcia","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"Sánchez","lastName":"Garcia","suffix":""},{"id":532783534,"identity":"3b9b1013-a079-4a0b-a4f4-bc81c0da7063","order_by":5,"name":"Mariano Martínez Marín","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"Mariano","middleName":"Martínez","lastName":"Marín","suffix":""},{"id":532783535,"identity":"462d31cb-575f-48a7-9560-0ed612e995bb","order_by":6,"name":"Javier David Benítez Fuentes","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"Javier","middleName":"David Benítez","lastName":"Fuentes","suffix":""},{"id":532783536,"identity":"01725ee8-ba79-44ae-b783-de4d3dd3f002","order_by":7,"name":"Isabel Chirivella González","email":"","orcid":"","institution":"Hospital Clínico Universitario de Valencia","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"Chirivella","lastName":"González","suffix":""},{"id":532783537,"identity":"6e6df4a4-3a72-4252-929b-000a9df2ac75","order_by":8,"name":"Ana Beatriz Sánchez Heras","email":"","orcid":"","institution":"Hospital General Universitario de Elche","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"Beatriz Sánchez","lastName":"Heras","suffix":""}],"badges":[],"createdAt":"2025-10-04 17:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7781365/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7781365/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":94140463,"identity":"d5534b61-3ab2-4446-9000-0f60b7298349","added_by":"auto","created_at":"2025-10-22 19:40:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":785352,"visible":true,"origin":"","legend":"","description":"","filename":"GenotypicandPhenotypicCharacteristicsofGermlineTP53VariantCarriersExperiencefromTwoCancerGeneticCounselingUnits.docx","url":"https://assets-eu.researchsquare.com/files/rs-7781365/v1/4021e8082b2da2202d6d9637.docx"},{"id":94139308,"identity":"9ec4b9cb-69d8-40dd-9f67-51a38d056a8d","added_by":"auto","created_at":"2025-10-22 19:32:50","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10478,"visible":true,"origin":"","legend":"","description":"","filename":"ba260e12b38b441ba9b085887d28f475.json","url":"https://assets-eu.researchsquare.com/files/rs-7781365/v1/38eeefe40803c4ec9f214ea0.json"},{"id":94139306,"identity":"2c942d4f-b516-4fe0-888b-c063c6d2d050","added_by":"auto","created_at":"2025-10-22 19:32:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21096,"visible":true,"origin":"","legend":"\u003cp\u003eTumor spectrum and multiplicity among germline \u003cem\u003eTP53\u003c/em\u003ecarriers. Distribution of first, second, and third malignant neoplasms across tumor types in the cohort (N=36).\u003cstrong\u003e \u003c/strong\u003eACC, adrenocortical carcinoma; CNS, central nervous system; LFS, Li–Fraumeni syndrome; MGP, multigene panel.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7781365/v1/af4a07ef22eac02020b42ff1.png"},{"id":94139309,"identity":"bf50be20-3a41-4fb2-820c-8389d498baae","added_by":"auto","created_at":"2025-10-22 19:32:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62698,"visible":true,"origin":"","legend":"\u003cp\u003eCatalogue of germline \u003cem\u003eTP53\u003c/em\u003e pathogenic/likely pathogenic variants. Variant cDNA/protein change, exon, type, functional class (Giacomelli/IARC), and number of carriers. DNE_LOF, dominant-negative with loss of function; IARC, International Agency for Research on Cancer; NA, not assessed; noDNE_LOF, non-dominant-negative with loss of function; noDNE_noLOF, non-dominant-negative without loss of function; PV, pathogenic/likely pathogenic variant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7781365/v1/83821e1d0c8e7602b300a54d.png"},{"id":94140462,"identity":"1a2546f8-699d-4baa-95b5-183419540b67","added_by":"auto","created_at":"2025-10-22 19:40:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35546,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional class, tumor distribution, and age at first cancer. Summary of numbers of variants, affected/unaffected carriers, tumor counts, and median age at first malignancy by functional class. ACC, adrenocortical carcinoma; DNE_LOF, dominant-negative with loss of function; IQR, interquartile range; LFS, Li–Fraumeni syndrome; NA, not assessed; noDNE_LOF, non-dominant-negative with loss of function; noDNE_noLOF, non-dominant-negative without loss of function; PV, pathogenic/likely pathogenic variant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7781365/v1/8698b808b35bcee64ff16eb9.png"},{"id":94140465,"identity":"962ee08c-62b9-4bb8-ba41-e7fcf6e7fca5","added_by":"auto","created_at":"2025-10-22 19:40:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":557473,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7781365/v1/598f02f2-086e-4cac-9cea-e0635cb8bb1f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genotypic and Phenotypic Characteristics of Germline TP53 Variant Carriers: Experience from Two Cancer Genetic Counseling Units","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLi\u0026ndash;Fraumeni syndrome (LFS) is an autosomal dominant cancer‑predisposition syndrome caused by germline pathogenic/likely pathogenic variants (P/LPVs) in \u003cem\u003eTP53\u003c/em\u003e, and is characterized by early‑onset \u0026ldquo;core\u0026rdquo; tumors\u0026mdash;sarcomas, premenopausal breast cancer \u0026le;31 years, adrenocortical carcinoma, and brain tumors.[1,2] Historically, ascertainment relied on clinical criteria (classic LFS; revised Chompret), which enriched for pediatric cancers and very early onset disease.[2] In contemporary practice, the widespread use of multigene panel (MGP) testing is uncovering \u003cem\u003eTP53\u003c/em\u003e carriers who do not meet traditional criteria, revealing a broader phenotypic spectrum with later ages at diagnosis and fewer core tumors than expected under classic LFS.[3,4,5]\u003c/p\u003e\n\u003cp\u003eBeyond clinical heterogeneity, \u003cem\u003eTP53\u003c/em\u003e variants are functionally diverse. The p53 protein is a central tumor suppressor that coordinates cell‑cycle arrest, apoptosis, senescence, and DNA damage responses.[6] Experimental models integrating dominant‑negative effect (DNE) and loss‑of‑function (LOF) have enabled functional grouping of germline variants (e.g., DNE_LOF, noDNE_LOF, noDNE_noLOF).[7] These groupings have been incorporated into curation frameworks and reference resources (ClinGen \u003cem\u003eTP53\u003c/em\u003e VCEP; IARC \u003cem\u003eTP53\u003c/em\u003e database) to improve risk interpretation.[8,9] Notably, some population variants\u0026mdash;such as the Brazilian founder p.R337H\u0026mdash;fall into non‑DNE classes and are linked to distinctive, often later‑onset patterns and tumor spectra.[10]\u003c/p\u003e\n\u003cp\u003eAgainst this backdrop, real‑world data are needed to clarify how ascertainment pathways and functional class relate to clinical expression. We report the tumor spectrum and age at first cancer in \u003cem\u003eTP53\u003c/em\u003e P/LPV carriers managed across two Spanish hereditary cancer units, comparing MGP‑ascertained carriers with those fulfilling clinical criteria, and testing whether DNE_LOF variants associate with earlier onset and core‑tumor enrichment relative to non‑DNE classes. Our goal is to provide concise, clinically actionable evidence to support risk stratification and individualized surveillance in \u003cem\u003eTP53\u003c/em\u003e‑related hereditary cancer.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eWe performed a retrospective cohort study including 36 carriers of germline \u003cem\u003eTP53\u003c/em\u003e P/LPVs from 18 unrelated families who attended two Spanish hereditary cancer units (Hospital General Universitario de Elche and Hospital Clínico Universitario de Valencia) between 2005 and 2023. Clinical data (demographics, personal and family cancer history, tumor dates and types, treatments, outcomes) were extracted from electronic medical records using a standardized template. The primary endpoint was age at first malignant neoplasm; secondary endpoints were tumor spectrum (first and subsequent primaries), frequency of “core” LFS tumors, intervals between successive primaries, and distributions by ascertainment pathway and functional class. The research question was whether age at onset and tumor spectrum differ (i) by ascertainment pathway and (ii) by functional class of the \u003cem\u003eTP53\u003c/em\u003e variant. We hypothesized that carriers identified through multigene panel (MGP) testing would present later and with fewer core tumors than those fulfilling clinical criteria, and that dominant-negative, loss-of-function (DNE_LOF) variants would be associated with earlier onset and enrichment for core tumors compared with non-dominant-negative, non-loss-of-function (noDNE_noLOF) variants.\u003c/p\u003e\n\u003cp\u003eInclusion required a documented germline \u003cem\u003eTP53\u003c/em\u003e P/LPV classified as pathogenic/likely pathogenic in ClinVar, either by one or more accredited clinical genetic testing laboratories or by the ClinGen \u003cem\u003eTP53\u003c/em\u003e Variant Curation Expert Panel (VCEP); variants of uncertain significance were excluded.[9,11] Carriers were grouped by ascertainment as: MGP testing in oncology; cascade testing for a known familial variant; clinical LFS (classic or 2015 revised Chompret criteria); or tumor-first detection with subsequent germline confirmation.[1,2] Core LFS tumors were defined as sarcoma, premenopausal breast cancer, adrenocortical carcinoma (ACC), and primary brain tumors.[1]\u003c/p\u003e\n\u003cp\u003eUnique variants were annotated by type and exon location and mapped to functional categories using the Giacomelli experimental model (integration of dominant-negative effect and loss-of-function) harmonized with IARC thresholds: DNE_LOF, noDNE_LOF, noDNE_noLOF; variants not evaluated in the assay were labeled “not assessed” (NA). The Brazilian founder variant p.R337H (c.1010G\u0026gt;A) was categorized as noDNE_noLOF.[7,8,9]\u003c/p\u003e\n\u003cp\u003eContinuous variables were summarized as medians (ranges or IQRs) and compared using two-sided Mann–Whitney U tests (α=0.05); categorical variables were compared with Fisher’s exact tests. For \u0026gt;2-group comparisons across functional classes, pairwise Mann–Whitney tests with Holm–Bonferroni correction were prespecified; where cell sizes were sparse, findings were summarized descriptively. Analyses were performed using IBM SPSS Statistics v28.\u003c/p\u003e\n\u003cp\u003eThis study was approved by the ethics committees of Hospital General Universitario de Elche and conducted in accordance with the principles of the Declaration of Helsinki. Given the exclusive use of de-identified data collected during routine care, a waiver of informed consent was granted.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eAmong the 36 carriers analyzed, women represented 58.3% and men 41.7%. Indications for genetic testing were cascade testing in 20 individuals (55.6%), MGP testing in 8 (22.2%), fulfillment of clinical LFS criteria in 7 (19.4%), and tumor-first detection with subsequent germline confirmation in 1 (2.8%). Of the MGP-ascertained carriers, seven were women with breast cancer and one was tested due to colonic polyposis.\u003c/p\u003e\n\u003cp\u003eOverall, 23/36 (63.9%) developed at least one malignant neoplasm (17 women, 6 men). The median age at first cancer was 36 years (range 27\u0026ndash;54); by sex, 37 (30\u0026ndash;52.5) for women and 30.5 (4\u0026ndash;54) for men. By ascertainment, median age at first cancer was 42 (37\u0026ndash;58) for MGP, 41 (33\u0026ndash;60.5) for cascade, and 27 (4\u0026ndash;30) for clinical LFS; the difference between MGP and clinical LFS was significant (\u003cem\u003ep\u003c/em\u003e = 0.011). First-tumor spectrum is summarized in \u003cstrong\u003eFigure (Fig.) 1\u003c/strong\u003e: breast cancer predominated (15/23, 65.2%; median 37 [30\u0026ndash;47] years), with two ACC, two brain tumors, and single cases of pancreatic, gastric, colorectal, and bladder cancers.\u003c/p\u003e\n\u003cp\u003eEight of the 23 affected carriers (34.7%) developed a second primary (7 women, 1 man), and four women (17.3%) developed a third primary (\u003cstrong\u003eFig. 1\u003c/strong\u003e). Among the eight with second primaries, 50% (4/8) were identified through MGP testing, 37.5% (3/8) met clinical criteria, and 12.5% (1/8) were tested due to a known familial variant. Breast cancer was the most common second primary (5/8, 62.5%); none had undergone prophylactic mastectomy prior to the second event. One second primary was a brain tumor and one a sarcoma (both LFS \u0026ldquo;core\u0026rdquo; tumors). No MGP-ascertained carrier developed a core tumor. The median interval between first and second primaries was 6 years (range 3\u0026ndash;9), and between second and third primaries 5.5 years (2\u0026ndash;9).\u003c/p\u003e\n\u003cp\u003eWe identified 15 distinct germline \u003cem\u003eTP53\u0026nbsp;\u003c/em\u003eP/LPVs; 12/15 (80%) were missense, with ~60% mapping to exons 5\u0026ndash;8. The Brazilian founder variant p.R337H (c.1010G\u0026gt;A) was present in four carriers from two families. The per-variant catalog with exon position, type, Giacomelli functional class, and number of carriers is shown in \u003cstrong\u003eFig. 2\u003c/strong\u003e, and class-level summaries are provided in \u003cstrong\u003eFig. 3\u003c/strong\u003e. Functional grouping comprised DNE_LOF in 6/15 (40%), noDNE_noLOF in 4/15 (26.6%), noDNE_LOF in 1/15 (6.6%), and not assessed (NA) in 4/15 (26.6%).\u003c/p\u003e\n\u003cp\u003eGenotype\u0026ndash;phenotype analyses (\u003cstrong\u003eFig. 3\u003c/strong\u003e) indicated earlier ages at first cancer for DNE_LOF (median 30 years [25\u0026ndash;38.5]) and NA (32 [27\u0026ndash;48]) compared with noDNE_noLOF (47 [37\u0026ndash;63]); the DNE_LOF versus noDNE_noLOF contrast was significant (\u003cem\u003ep\u003c/em\u003e = 0.034). Among women with breast cancer, earlier ages were observed for DNE_LOF (median 34 [28.5\u0026ndash;49.5]) and NA (31 [27\u0026ndash;37]) compared with noDNE_noLOF (46.5 [37\u0026ndash;63]). Core tumors were enriched among DNE_LOF carriers, with exceptions of one sarcoma in NA and one ACC in a p.R337H carrier. Among those with second primaries, 4/8 (50%) were DNE_LOF, 2/8 (25%) noDNE_noLOF, and 2/8 (25%) NA; third primaries occurred in 2/4 DNE_LOF and 2/4 NA carriers. Within the MGP subset, 50% of variants were noDNE_noLOF, consistent with the older age at onset and milder spectrum in that group.\u003c/p\u003e\n\u003cp\u003eAt last follow-up, 8/23 (34.7%) affected carriers had died (median age at death 56 years [42\u0026ndash;62]); two deaths were non-cancer (stroke).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur study adds evidence that clinical expression among germline \u003cem\u003eTP53\u003c/em\u003e carriers varies with both variant function and ascertainment. In our series, individuals identified by MGP testing presented later and did not develop core LFS tumours, a pattern consistent with panel‑based cohorts in which carriers are older at diagnosis and less likely to satisfy classic criteria [3]. Conversely, carriers from clinically ascertained LFS families showed earlier onset and more core tumours, echoing previous observations [12,13]. Taken together, these findings support a spectrum of heritable \u003cem\u003eTP53\u003c/em\u003e‑related cancer risk in which genotype and ascertainment inform comprehensive surveillance throughout adulthood.\u003c/p\u003e\n\u003cp\u003eDifferences in variant function account for much of the heterogeneity seen in germline \u003cem\u003eTP53\u003c/em\u003e. This pattern is consistent with foundational functional maps. \u003cem\u003eKato et al.\u003c/em\u003e performed a high‑throughput yeast transactivation assay covering thousands of single‑amino‑acid substitutions and showed that many DNA‑binding domain missense variants have severe loss of transcriptional activity and dominant‑negative behavior [14]. \u003cem\u003eKotler et al.\u003c/em\u003e used deep mutational scanning in mammalian cells to survey near‑complete missense space and linked measured functional impact to human tumor hotspot frequencies [15]. Other works further show that mutant p53 can drive genomic instability and acquire oncogenic gain‑of‑function properties beyond simple haploinsufficiency [16,17]. At the clinical level, a large observational cohort reported that LOF (with or without DNE) variants were associated with significantly earlier age at first cancer than variants retaining transactivation, reinforcing a functional gradient of penetrance [5]. Complementing this, a retrospective, panel‑based series found that panel‑ascertained \u003cem\u003eTP53\u003c/em\u003e carriers were older at first diagnosis and less likely to meet classic LFS criteria than clinically ascertained families [3]. Our data mirror these trends, none of the MGP‑ascertained carriers developed core tumors, and their first cancers occurred later, while DNE_LOF versus noDNE_noLOF showed a 17‑year median age difference (\u003cem\u003ep\u003c/em\u003e=0.034). Variants without assay data in our study behaved more like DNE_LOF than noDNE_noLOF—similar to other disruptive alleles in large cohorts—but expert reviews caution that functional class should guide, not determine, management until prospective, variant-stratified data are available [18].\u003c/p\u003e\n\u003cp\u003eThe Brazilian founder p.R337H illustrates how a single allele can shape population‑level risk. Achatz et al. first characterized family‑based series from Southern/Southeastern Brazil showing that \u003cem\u003ep\u003c/em\u003e.R337H segregates with LFS and LFS-like phenotypes yet often with later adult onset than canonical DNA‑binding mutations [19]. A landmark statewide neonatal screening and surveillance program demonstrated that identifying \u003cem\u003ep\u003c/em\u003e.R337H carriers at birth facilitated early‑stage detection of pediatric ACC and improved outcomes compared with historical controls [20]. Broader institutional cohorts confirm that \u003cem\u003ep\u003c/em\u003e.R337H families manifest a bimodal pattern—substantial risk for childhood ACC plus adult cancers (e.g., breast, brain, sarcoma) that tend to arise later than in classic LFS, with some series noting higher frequencies of thyroid or renal cancers [10,21]. Our experience was concordant: two of four \u003cem\u003ep\u003c/em\u003e.R337H carriers developed malignancies (ACC at 1 year; breast cancer at 36 years), showing the need for variant‑aware counseling (e.g., pediatric adrenal screening) layered onto core LFS surveillance.\u003c/p\u003e\n\u003cp\u003eConsensus statements recommend annual whole‑body MRI (WBMRI), periodic brain MRI, targeted organ imaging, and age‑appropriate biochemical screening from childhood onward, with adaptation to local resources [22,23]. Evidence for benefit comes from prospective observational studies by Villani et al.: the initial report and 11‑year update showed that structured surveillance detects more asymptomatic, early‑stage tumors and is associated with markedly higher 5‑year overall survival (~89% under surveillance vs ~60% in those declining) compared with historical/unscreened comparators [12,13]. Therapeutic decisions also benefit from a \u003cem\u003eTP53\u003c/em\u003e‑specific lens. A consensus review (Thariat et al.) and a multi‑institution retrospective series (Hendrickson et al.) suggest that when clinically equivalent alternatives exist, radiotherapy (RT) should be avoided or adapted to reduce radiation‑associated second primaries; if RT is necessary, dose/fields should be minimized with vigilant follow‑up [24,25]. For breast cancer—the most common first malignancy in our study—a large analysis reported that \u003cem\u003eTP53\u003c/em\u003e‑associated tumors are often high‑grade and HER2‑positive, supporting early MRI‑based screening and, in selected carriers, discussion of bilateral risk‑reducing mastectomy to avoid adjuvant RT and lower contralateral risk [26].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAlongside these genotype–phenotype observations, two interpretive caveats remain central when applying TP53 testing in clinical settings. Low-allele-fraction TP53 calls in blood can reflect clonal hematopoiesis or post-zygotic mosaicism rather than true germline, and several clinical series have shown that orthogonal testing in non-hematopoietic tissue (such as fibroblasts or saliva) is essential to avoid misclassifying somatic events as heritable LFS [27,28].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main limitations of this study include its retrospective design, small sample size, and potential referral bias, which limit statistical power and generalizability. Reliance on electronic medical records may have introduced variability in the level of clinical detail, and germline status was inferred from blood only without orthogonal confirmation, while some variants could not be functionally classified. Despite these limitations, the study has strengths: it draws on two hereditary cancer units, combines carriers identified through both clinical criteria and multigene panel testing, and applies functional classification to explore genotype–phenotype correlations. These features provide useful real-world insights and support the need for larger, prospective, standardized studies to validate and extend our findings.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study shows that clinical expression among germline \u003cem\u003eTP53\u003c/em\u003e P/LPV carriers is influenced by both functional class and ascertainment pathway. DNE_LOF and NA variants were associated with earlier onset and a higher frequency of core LFS tumors, whereas carriers identified through MGP testing, often with noDNE_noLOF variants, presented later and with milder phenotypes. These findings emphasize the heterogeneity of \u003cem\u003eTP53\u003c/em\u003e-related hereditary cancer and support incorporating functional classification into risk assessment to better tailor surveillance and counseling strategies.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSubmitting Declarations:\u0026nbsp;\u003c/strong\u003eWe confirm that the manuscript is original, has not been published previously, and is not under consideration elsewhere. All authors have read and approved the final version of the manuscript, and declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors received no financial support for the research, authorship, and/or publication of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eS\u0026aacute;nchez-Heras AB, Ramon Y Cajal T, Pineda M, et al. SEOM clinical guideline on heritable TP53-related cancer syndrome (2022). \u003cem\u003eClin Transl Oncol\u003c/em\u003e. 2023;25(9):2627-2633. doi:10.1007/s12094-023-03202-9\u003c/li\u003e\n\u003cli\u003eBougeard G, Renaux-Petel M, Flaman JM, et al. Revisiting Li-Fraumeni Syndrome From TP53 Mutation Carriers. \u003cem\u003eJ Clin Oncol\u003c/em\u003e. 2015;33(21):2345-2352. doi:10.1200/JCO.2014.59.5728\u003c/li\u003e\n\u003cli\u003eRana HQ, Gelman R, LaDuca H, et al. Differences in TP53 Mutation Carrier Phenotypes Emerge From Panel-Based Testing. \u003cem\u003eJ Natl Cancer Inst\u003c/em\u003e. 2018;110(8):863-870. doi:10.1093/jnci/djy001\u003c/li\u003e\n\u003cli\u003eRana HQ, Clifford J, Hoang L, et al. Genotype-phenotype associations among panel-based TP53+ subjects. \u003cem\u003eGenet Med\u003c/em\u003e. 2019;21(11):2478-2484. doi:10.1038/s41436-019-0541-y\u003c/li\u003e\n\u003cli\u003ede Andrade KC, Khincha PP, Hatton JN, et al. Cancer incidence, patterns, and genotype-phenotype associations in individuals with pathogenic or likely pathogenic germline TP53 variants: an observational cohort study. \u003cem\u003eLancet Oncol\u003c/em\u003e. 2021;22(12):1787-1798. doi:10.1016/S1470-2045(21)00580-5\u003c/li\u003e\n\u003cli\u003eOlivier M, Hollstein M, Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use. \u003cem\u003eCold Spring Harb Perspect Biol\u003c/em\u003e. 2010;2(1):a001008. doi:10.1101/cshperspect.a001008\u003c/li\u003e\n\u003cli\u003eGiacomelli AO, Yang X, Lintner RE, et al. Mutational processes shape the landscape of TP53 mutations in human cancer. \u003cem\u003eNat Genet\u003c/em\u003e. 2018;50(10):1381-1387. doi:10.1038/s41588-018-0204-y\u003c/li\u003e\n\u003cli\u003eBouaoun L, Sonkin D, Ardin M, et al. TP53 Variations in Human Cancers: New Lessons from the IARC TP53 Database and Genomics Data. \u003cem\u003eHum Mutat\u003c/em\u003e. 2016;37(9):865-876. doi:10.1002/humu.23035\u003c/li\u003e\n\u003cli\u003eFortuno C, Lee K, Olivier M, et al. Specifications of the ACMG/AMP variant interpretation guidelines for germline TP53 variants. \u003cem\u003eHum Mutat\u003c/em\u003e. 2021;42(3):223-236. doi:10.1002/humu.24152\u003c/li\u003e\n\u003cli\u003eFerreira AM, Brondani VB, Helena VP, et al. Clinical spectrum of Li-Fraumeni syndrome/Li-Fraumeni-like syndrome in Brazilian individuals with the TP53 p.R337H mutation. \u003cem\u003eJ Steroid Biochem Mol Biol\u003c/em\u003e. 2019;190:250-255. doi:10.1016/j.jsbmb.2019.04.011\u003c/li\u003e\n\u003cli\u003eLandrum MJ, Lee JM, Benson M, et al. ClinVar: improving access to variant interpretations and supporting evidence. \u003cem\u003eNucleic Acids Res\u003c/em\u003e. 2018;46(D1):D1062-D1067. doi:10.1093/nar/gkx1153\u003c/li\u003e\n\u003cli\u003eVillani A, Tabori U, Schiffman J, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: a prospective observational study. \u003cem\u003eLancet Oncol\u003c/em\u003e. 2011;12(6):559-567. doi:10.1016/S1470-2045(11)70119-X\u003c/li\u003e\n\u003cli\u003eVillani A, Shore A, Wasserman JD, et al. Biochemical and imaging surveillance in germline TP53 mutation carriers with Li-Fraumeni syndrome: 11 year follow-up of a prospective observational study. \u003cem\u003eLancet Oncol\u003c/em\u003e. 2016;17(9):1295-1305. doi:10.1016/S1470-2045(16)30249-2\u003c/li\u003e\n\u003cli\u003eKato S, Han SY, Liu W, et al. Understanding the function-structure and function-mutation relationships of p53 tumor suppressor protein by high-resolution missense mutation analysis. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e. 2003;100(14):8424-8429. doi:10.1073/pnas.1431692100\u003c/li\u003e\n\u003cli\u003eKotler E, Shani O, Goldfeld G, et al. A Systematic p53 Mutation Library Links Differential Functional Impact to Cancer Mutation Pattern and Evolutionary Conservation. \u003cem\u003eMol Cell\u003c/em\u003e. 2018;71(1):178-190.e8. doi:10.1016/j.molcel.2018.06.012\u003c/li\u003e\n\u003cli\u003eZerdoumi Y, Lanos R, Raad S, et al. Germline TP53 mutations result into a constitutive defect of p53 DNA binding and transcriptional response to DNA damage. \u003cem\u003eHum Mol Genet\u003c/em\u003e. 2017;26(14):2591-2602. doi:10.1093/hmg/ddx106\u003c/li\u003e\n\u003cli\u003eMuller PA, Vousden KH. Mutant p53 in cancer: new functions and therapeutic opportunities. \u003cem\u003eCancer Cell\u003c/em\u003e. 2014;25(3):304-317. doi:10.1016/j.ccr.2014.01.021\u003c/li\u003e\n\u003cli\u003eFortuno C, Pesaran T, Mester J, et al. Genotype-phenotype correlations among TP53 carriers: Literature review and analysis of probands undergoing multi-gene panel testing and single-gene testing. \u003cem\u003eCancer Genet\u003c/em\u003e. 2020;248-249:11-17. doi:10.1016/j.cancergen.2020.09.002\u003c/li\u003e\n\u003cli\u003eAchatz MI, Olivier M, Le Calvez F, et al. The TP53 mutation, R337H, is associated with Li-Fraumeni and Li-Fraumeni-like syndromes in Brazilian families. \u003cem\u003eCancer Lett\u003c/em\u003e. 2007;245(1-2):96-102. doi:10.1016/j.canlet.2005.12.039\u003c/li\u003e\n\u003cli\u003eCust\u0026oacute;dio G, Parise GA, Kiesel Filho N, et al. Impact of neonatal screening and surveillance for the TP53 R337H mutation on early detection of childhood adrenocortical tumors. \u003cem\u003eJ Clin Oncol\u003c/em\u003e. 2013;31(20):2619-2626. doi:10.1200/JCO.2012.46.3711\u003c/li\u003e\n\u003cli\u003eFormiga MNDC, de Andrade KC, Kowalski LP, Achatz MI. Frequency of Thyroid Carcinoma in Brazilian TP53 p.R337H Carriers With Li Fraumeni Syndrome. JAMA Oncol. 2017;3(10):1400-1402. doi:10.1001/jamaoncol.2016.6389\u003c/li\u003e\n\u003cli\u003eKratz CP, Achatz MI, Brugi\u0026egrave;res L, et al. Cancer Screening Recommendations for Individuals with Li-Fraumeni Syndrome. \u003cem\u003eClin Cancer Res\u003c/em\u003e. 2017;23(11):e38-e45. doi:10.1158/1078-0432.CCR-17-0408\u003c/li\u003e\n\u003cli\u003eFrebourg T, Bajalica Lagercrantz S, Oliveira C, Magenheim R, Evans DG; European Reference Network GENTURIS. Guidelines for the Li-Fraumeni and heritable TP53-related cancer syndromes. Eur J Hum Genet. 2020;28(10):1379-1386. doi:10.1038/s41431-020-0638-4\u003c/li\u003e\n\u003cli\u003eThariat J, Chevalier F, Orbach D, et al. Avoidance or adaptation of radiotherapy in patients with cancer with Li-Fraumeni and heritable TP53-related cancer syndromes. Lancet Oncol. 2021;22(12):e562-e574. doi:10.1016/S1470-2045(21)00425-3\u003c/li\u003e\n\u003cli\u003eHendrickson PG, Luo Y, Kohlmann W, et al. Radiation therapy and secondary malignancy in Li-Fraumeni syndrome: A hereditary cancer registry study. Cancer Med. 2020;9(21):7954-7963. doi:10.1002/cam4.3427\u003c/li\u003e\n\u003cli\u003eBreast Cancer Association Consortium, Mavaddat N, Dorling L, et al. Pathology of Tumors Associated With Pathogenic Germline Variants in 9 Breast Cancer Susceptibility Genes. \u003cem\u003eJAMA Oncol\u003c/em\u003e. 2022;8(3):e216744. doi:10.1001/jamaoncol.2021.6744\u003c/li\u003e\n\u003cli\u003eWeitzel JN, Chao EC, Nehoray B, et al. Somatic TP53 variants frequently confound germ-line testing results. Genet Med. 2018;20(8):809-816. doi:10.1038/gim.2017.196\u003c/li\u003e\n\u003cli\u003eMester JL, Jackson SA, Postula K, et al. Apparently Heterozygous TP53 Pathogenic Variants May Be Blood Limited in Patients Undergoing Hereditary Cancer Panel Testing. J Mol Diagn. 2020;22(3):396-404. doi:10.1016/j.jmoldx.2019.12.003\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003e\u0026nbsp;\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"familial-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fame","sideBox":"Learn more about [Familial Cancer](http://link.springer.com/journal/10689)","snPcode":"10689","submissionUrl":"https://submission.nature.com/new-submission/10689/3","title":"Familial Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TP53, Li–Fraumeni, Germline variants, Hereditary cancer, Genotype–phenotype correlation, Multigene panel testing","lastPublishedDoi":"10.21203/rs.3.rs-7781365/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7781365/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Germline TP53 pathogenic or likely pathogenic variants underlie Li–Fraumeni syndrome (LFS), but with broader use of multigene panel (MGP) testing carriers are increasingly identified outside classic criteria. We retrospectively studied 36 carriers from 18 families evaluated at two Spanish hereditary cancer units (2005–2023). Clinical data were obtained from medical records, and variants were functionally classified using the Giacomelli model and IARC thresholds as dominant-negative with loss of function (DNE_LOF), non-dominant-negative without loss of function (noDNE_noLOF), non-dominant-negative with loss of function, or not assessed. Descriptive statistics summarized the tumor spectrum and group comparisons were performed with the Mann–Whitney U test (α=0.05). Overall, 23 of 36 carriers (63.9%) developed at least one malignancy; 58.3% were female and the median age at first cancer was 36 years (range 27–54). Patients identified through MGP testing had later onset compared with those meeting clinical LFS criteria (median 42 [37–58] vs 27 [4–30] years; p=0.011). Breast cancer was the most frequent tumor (65% of first cancers, median age 37 [30–47]), whereas none of the MGP-ascertained carriers developed LFS “core” tumors (sarcoma, brain tumor, adrenocortical carcinoma). Fifteen distinct germline variants were identified; 80% were missense, mainly in exons 5–8. DNE_LOF (median 30 [25–38.5]) and not-assessed variants (32 [27–48]) were associated with earlier onset compared with noDNE_noLOF (47 [37–63]; p=0.034). Eight carriers (34.7%) developed second primaries after a median interval of 6 years, and four developed third primaries. Functional classification stratifies risk, supporting a broader TP53-related hereditary cancer syndrome and individualized surveillance.","manuscriptTitle":"Genotypic and Phenotypic Characteristics of Germline TP53 Variant Carriers: Experience from Two Cancer Genetic Counseling Units","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-22 19:32:46","doi":"10.21203/rs.3.rs-7781365/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T09:01:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-26T21:28:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"264668008527088292977126232542666588413","date":"2025-10-09T09:31:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-09T08:40:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-08T13:31:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-08T13:31:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Familial Cancer","date":"2025-10-04T17:43:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"familial-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fame","sideBox":"Learn more about [Familial Cancer](http://link.springer.com/journal/10689)","snPcode":"10689","submissionUrl":"https://submission.nature.com/new-submission/10689/3","title":"Familial Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"99296be6-b30d-40a2-a4a9-6db536eece56","owner":[],"postedDate":"October 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T08:59:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-22 19:32:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7781365","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7781365","identity":"rs-7781365","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}