A quantitative, Bayesian-informed approach to gene-specific variant classification: Updated Expert Panel recommendations improve classification of TP53 germline variants for Li-Fraumeni syndrome

Spectrum, molecular features, and clinical outcomes of hematologic malignancies and clonal hematopoiesis in li fraumeni syndrome

Evolution of germline TP53 variant classification in children with cancer

TP53 Germline Variations Influence the Predisposition and Prognosis of Acute Lymphoblastic Leukemia in Children

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome with exceptionally high lifetime cancer risks, caused primarily by germline pathogenic variants in TP53. Sarcomas, pre-menopausal breast cancer, brain tumors and adrenocortical carcinomas are the ‘core’ LFS cancers among a multitude of other cancers that can occur. Individuals with LFS are also at risk of developing multiple primary cancers over their lifetime. Clinical criteria have been established to denote LFS and ‘Li-Fraumeni like’ (LFL) families to facilitate genetic testing for TP53. De novo pathogenic germline variants in TP53 are estimated to cause between 7-25% of LFS. Gonadal mosaicism is an alternate explanation for presumed de novo variants and is yet to be described in LFS. We present a proband who developed WHO grade II astrocytoma at age 18 years, who had a deceased brother with osteosarcoma diagnosed at age 13 years, and a healthy older sibling. The only other cancer history was leukemia at age 62 years in the paternal grandmother, who also had polycythemia vera. The family meets Eele’s criteria for LFL. The proband’s germline genetic testing revealed a heterozygous TP53 c.743G>A p.R248Q pathogenic variant. Subsequent clinical genetic testing of the parents failed to reveal the variant, and the proband was presumed to have a de novo variant. Given the characteristic LFS-associated cancers in the brothers, we investigated further and identified the same TP53 pathogenic variant in the brother’s osteosarcoma tissue and pathology-confirmed benign liver tissue. TP53 sequencing of the brother’s tumor did not show loss of heterozygosity. Parental relationships to the proband and brother were confirmed through STR-based identifiler profiling. Ultra-deep sequencing (>50,000x) of DNA derived from the parents’ blood, saliva and father’s benign colon polyp did not identify the variant, thus ruling out somatic mosaicism in the parents. We were unable to test gonadal tissues from the parents. In the setting of two siblings with the same pathogenic variant in TP53 which is absent in both parents, this family presents a case of assumed gonadal mosaicism resulting from a post-zygotic event. The possibility of gonadal mosaicism presents complexity in genetic counselling of families with inherited disorders, and a conundrum in syndromes such as TP53-associated LFS where the presumed de novo rate is high and cancer risks are significantly elevated. The presented family suggests that further investigation of individuals with presumed de novo variation, especially in the setting of suspicious family cancer history, is required to accurately identify family members at-risk. Citation Format: Payal P. Khincha, Kristine Jones, Kedest Teshome, Belynda Hicks, Phuong L. Mai, Sharon A. Savage. Gonadal mosaicism in a family with TP53-associated Li-Fraumeni syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4159.

We appreciate the Letter to the Editor by Evans, Turnbull, and Woodward (2019) on our manuscript “Variable population prevalence estimates of germline TP53 variants: A gnomAD-based analysis” (de Andrade et al., 2019) and would like to take this opportunity to address key aspects of the letter. We agree with the statement by Evans et al. (2019) that clinical considerations related to germline TP53 variant classification are of critical importance. Our article (de Andrade et al., 2019) aimed to conduct a population-level approach to estimate the prevalence of germline TP53 variants in a publicly available database of presumed non-cancer individuals, with its inherent biases and challenges. In our manuscript, nonclinical classification criteria were established for research-purposes to allow for the discovery of potentially deleterious variants. These criteria were not intended for application in the clinical setting of germline TP53 variant curation, nor were they intended to estimate the prevalence of Li-Fraumeni syndrome. We acknowledged this in the Methods section, as follows: “Importantly, these nonclinical classification criteria are independent of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG-AMP) guidelines for variant classification (Richards et al., 2015). Therefore, these criteria are not necessarily representative of the more stringent methods utilized when determining the pathogenicity of a variant for clinical use.” (de Andrade et al., 2019). Variant classification is a dynamic process that is dependent on the emergence of novel functional data, bioinformatic prediction tool advancements, and clinical data sharing. This evolving process may ultimately reclassify certain variants, leading to potential changes in the estimated population prevalence of pathogenic/likely pathogenic germline TP53 variants. Robust variant classification combined with clinical criteria is required for a proper diagnosis of Li-Fraumeni syndrome. We would like to highlight that the prevalence of Li-Fraumeni syndrome cannot be determined by the population frequency of pathogenic germline TP53 variants alone. Li-Fraumeni syndrome is a clinical diagnosis on the basis of personal and family history of cancer. As discussed in our manuscript and reinforced herein, germline TP53 variant curation in relation to Li-Fraumeni syndrome should be comprehensively evaluated on the basis of family history, clinical standards, and established clinical variant classification guidelines (Amendola et al., 2016; Richards et al., 2015). Clinical genetic testing, cancer surveillance, and management recommendations for Li-Fraumeni syndrome-associated TP53 variants must be made by specialists after thorough consideration of the individual cancer risk. The authors declare that there are no conflict of interests.

Pathogenic germline variants in TP53 predispose carriers to the multi-cancer Li-Fraumeni syndrome (LFS). Widespread multigene panel testing is identifying TP53 pathogenic variants in breast cancer patients outside the strict clinical criteria recommended for LFS testing. We aimed to assess frequency and clinical implications of TP53 pathogenic variants in breast cancer cohorts ascertained outside LFS. Classification of TP53 germline variants reported in 59 breast cancer studies, and publicly available population control sets was reviewed and identified evidence for misclassification of variants. TP53 pathogenic variant frequency was determined for: breast cancer studies grouped by ascertainment characteristics; breast cancer cohorts undergoing panel testing; and population controls. Early age of breast cancer onset, regardless of family history or BRCA1/BRCA2 previous testing, had the highest pick-up rate for TP53 carriers. Patients at risk of hereditary breast cancer unselected for features of LFS carried TP53 pathogenic variants at a frequency comparable to that of other non-BRCA1/2 breast cancer predisposing genes, and ∼threefold more than reported in population controls. These results have implications for the implementation of TP53 testing in broader clinical settings, and suggest urgent need to investigate cancer risks associated with TP53 pathogenic variants in individuals outside the LFS spectrum.

Germline pathogenic variants in TP53 are associated with Li-Fraumeni syndrome, a cancer predisposition disorder inherited in an autosomal dominant pattern associated with a high risk of malignancy, including early-onset breast cancers, sarcomas, adrenocortical carcinomas, and brain tumors. Intense cancer surveillance for individuals with TP53 germline pathogenic variants is associated with reduced cancer-related mortality. Accurate and consistent classification of germline variants across clinical and research laboratories is important to ensure appropriate cancer surveillance recommendations. Here, we describe the work performed by the Clinical Genome Resource TP53 Variant Curation Expert Panel (ClinGen TP53 VCEP) focused on specifying the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines for germline variant classification to the TP53 gene. Specifications were developed for 20 ACMG/AMP criteria, while nine were deemed not applicable. The original strength level for the 10 criteria was also adjusted due to current evidence. Use of TP53-specific guidelines and sharing of clinical data amongexperts and clinical laboratories led to a decrease in variants of uncertain significance from 28% to 12% compared with the original guidelines. The ClinGen TP53 VCEP recommends the use of these TP53-specific ACMG/AMP guidelines as the standard strategy for TP53 germline variant classification.

Pathogenic variants in TP53 have been classically thought to cause Li-Fraumeni syndrome (LFS), a cancer predisposition with high risks for various childhood- and adult-onset malignancies. However, increased genetic testing has lately revealed, that pathogenic variant carriers exhibit a broader range of phenotypes and that penetrance may be dependent both on variant type and modifiers. Using next generation sequencing and short tandem repeat analysis, we identified germline pathogenic variants in TP53 and RAD51C located in cis on chromosome 17 in a 43-year-old male, who has developed a rare sebaceous gland carcinoma (SGC) but so far no tumors of the LFS spectrum. This course mirrors a Trp53-Rad51c-double-mutant cis mouse-model, which similarly develops SGC, while the characteristic Trp53-associated tumor spectrum occurs with significantly lower frequency. Therefore, we propose that co-occurent pathogenic variants in RAD51C and TP53 may predispose to SGC, reminiscent of Muir-Torre syndrome. Further, this report supports the diversity of clinical presentations associated with germline TP53 alterations, and thus, the proposed expansion of LFS to heritable TP53-related cancer syndrome.

Reports of variable cancer penetrance in Li-Fraumeni syndrome (LFS) have raised questions regarding the prevalence of pathogenic germline TP53 variants. We previously reported higher-than-expected population prevalence estimates in sequencing databases composed of individuals unselected for cancer history. This study aimed to expand and further evaluate the prevalence of pathogenic and likely pathogenic germline TP53 variants in the gnomAD dataset (version r2.0.2, n=138,632). Variants were selected and classified based on our previously published algorithm and compared with alternative estimates based on three different classification databases: ClinVar, HGMD, and the UMD_TP53 database. Conservative prevalence estimates of pathogenic and likely pathogenic TP53 variants were within the range of one carrier in 3,555-5,476 individuals. Less stringent classification increased the approximate prevalence to one carrier in every 400-865 individuals, mainly due to the inclusion of the controvertible p.N235S, p.V31I, and p.R290H variants. This study shows a higher-than-expected population prevalence of pathogenic and likely pathogenic germline TP53 variants even with the most conservative estimates. However, these estimates may not necessarily reflect the prevalence of the classical LFS phenotype, which is based upon family history of cancer. Comprehensive approaches are needed to better understand the interplay of germline TP53 variant classification, prevalence estimates, cancer penetrance, and LFS-associated phenotype.

Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disorder characterized by an extreme lifetime risk of multiple and early-onset tumors, driven by inherited pathogenic variants in the TP53 gene. While somatic mutations in TP53 are among the most frequent genetic alterations in cancer, germline mutations remain rare. Although LFS has long been recognized as a prototypical cancer predisposition syndrome, recent advances have significantly reshaped its diagnostic criteria and deepened our understanding of its associated cancer risks. The review illustrates the evolving diagnostic landscape of LFS and the updated indication criteria for germline TP53 testing, which have broadened the definition of the syndrome into the more inclusive entity of heritable TP53-related cancer predisposition syndrome (hTP53rc). The adoption of NGS has streamlined molecular diagnostics in hereditary cancer syndromes. Germline analysis of TP53 has become standard practice in hereditary cancer predisposition testing, even if a proband does not exhibit a LFS phenotype. However, the accurate identification of germline pathogenic TP53 variants remains challenging, particularly due to confounding factors, such as mosaicism or clonal hematopoiesis of indeterminate potential. Confirmatory testing using an independent tissue sample, along with estimation of allelic fraction is necessary to distinguish true germline variants. Another major hurdle is the assessment of the pathogenicity of rare germline TP53 variants, which requires a thorough genotype-phenotype correlation analyses. Recently, gene-specific American College of Medical Genetics and Genomics / Association for Molecular Pathology criteria have been introduced to support the classification of germline TP53 variants. Importantly, only carriers of a clearly established germline pathogenic variant should be considered for inclusion in an intensive clinical surveillance and prevention program. The present work underscores a paradigm shift in the understanding of one of the most significant cancer predisposition syndromes and aims to stimulate further discussion on the organization of care for high-risk carriers of pathogenic TP53 variants in the Czech Republic.

Genome-first approach of the prevalence and cancer phenotypes of pathogenic or likely pathogenic germline TP53 variants

Li-Fraumeni syndrome (LFS) is an inherited, autosomal-dominant condition that predisposes individuals to a wide-spectrum of tumors at an early age. Approximately 70% of families with classic LFS have pathogenic variants in the tumor suppressor gene TP53 that disrupt protein function or stability. While more than 70% of pathogenic variants in TP53 are missense variants, the vast majority occur very infrequently, and thus their clinical significance is uncertain or conflicting. Here, we report an extremely rare TP53 missense variant, c.799C > T (p.Arg267Trp), identified in a 2-year-old Saudi proband diagnosed with choroid plexus carcinoma (CPC) and six of his first- and second-degree relatives. CPC is frequently found in families with LFS, and this is the first detailed report of a family with this variant. Intriguingly, the proband’s father is homozygous for TP53 c.799C > T and phenotypically normal at 39 years of age. While loss of TP53 heterozygosity is often observed in tumors from individuals with LFS, homozygous germline TP53 pathogenic variants are rare. Based on our analysis of this single family, we hypothesize that TP53 c.799C > T has low or variable penetrance for LFS, with predisposition to the development of CPC. The observations from this family have furthered our understanding of the phenotypic variability that may be caused by one variant of TP53, even in the same family, and suggest that other factors (genetic and/or environmental) may play a role in mechanism of disease manifestation in LFS.

Li-Fraumeni syndrome is a cancer predisposition syndrome that is associated with a high, lifelong risk of a broad spectrum of cancers that is caused by pathogenic TP53 germline variants. A definition that reflects the broad phenotypic spectrum that has evolved since the gene discovery is lacking, and mechanisms leading to phenotypic differences remain largely unknown. To define the phenotypic spectrum of Li-Fraumeni syndrome and conduct phenotype-genotype associations across the phenotypic spectrum. We analyzed and classified the germline variant data set of the International Agency for Research on Cancer TP53 database that contains data on a cohort of 3034 persons from 1282 families reported in the scientific literature since 1990. We defined the term Li-Fraumeni spectrum to encompass (1) phenotypic Li-Fraumeni syndrome, defined by the absence of a pathogenic/likely pathogenic TP53 variant in persons/families meeting clinical Li-Fraumeni syndrome criteria; (2) Li-Fraumeni syndrome, defined by the presence of a pathogenic/likely pathogenic TP53 variant in persons/families meeting Li-Fraumeni syndrome testing criteria; (3) attenuated Li-Fraumeni syndrome, defined by the presence of a pathogenic/likely pathogenic TP53 variant in a person/family with cancer who does not meet Li-Fraumeni syndrome testing criteria; and (4) incidental Li-Fraumeni syndrome, defined by the presence of a pathogenic/likely pathogenic TP53 variant in a person/family without a history of cancer. Data analysis occurred from November 2020 to March 2021. Differences in variant distribution and cancer characteristics in patients with a germline TP53 variant who met vs did not meet Li-Fraumeni syndrome testing criteria. Tumor spectra showed significant differences, with more early adrenal (n = 166, 6.5% vs n = 0), brain (n = 360, 14.17% vs n = 57, 7.46%), connective tissue (n = 303, 11.92% vs n = 56, 7.33%), and bone tumors (n = 279, 10.98% vs n = 3, 0.39%) in patients who met Li-Fraumeni syndrome genetic testing criteria (n = 2139). Carriers who did not meet Li-Fraumeni syndrome genetic testing criteria (n = 678) had more breast (n = 292, 38.22% vs n = 700, 27.55%) and other cancers, 45% of them occurring after age 45 years. Hotspot variants were present in both groups. Several variants were exclusively found in patients with Li-Fraumeni syndrome, while others where exclusively found in patients with attenuated Li-Fraumeni syndrome. In patients who met Li-Fraumeni syndrome genetic testing criteria, most TP53 variants were classified as pathogenic/likely pathogenic (1757 of 2139, 82.2%), whereas 40.4% (404 of 678) of TP53 variants identified in patients who did not meet the Li-Fraumeni syndrome genetic testing criteria were classified as variants of uncertain significance, conflicting results, likely benign, benign, or unknown. The findings of this cohort study suggest that this new classification, Li-Fraumeni spectrum, is a step toward understanding the factors that lead to phenotypic differences and may serve as a model for other cancer predisposition syndromes.

Genetic testing for Li-Fraumeni syndrome (LFS) is performed by using blood specimens from patients selected based on phenotype-dependent guidelines. This approach is problematic for understanding the LFS clinical spectrum because patients with nonclassical presentations are missed, clonal hematopoiesis-related somatic blood alterations cannot be distinguished from germline variants, and unrelated tumors cannot be differentiated from those driven by germline TP53 defects. To provide insights into the LFS-related cancer spectrum, we analyzed paired tumor-blood DNA sequencing results in 17 922 patients with cancer and distinguished clonal hematopoiesis-related, mosaic, and germline TP53 variants. Loss of heterozygosity and TP53 mutational status were assessed in tumors, followed by immunohistochemistry for p53 expression on a subset to identify those lacking biallelic TP53 inactivation. Pathogenic/likely pathogenic TP53 variants were identified in 50 patients, 12 (24.0%) of which were clonal hematopoiesis related and 4 (8.0%) of which were mosaic. Twelve (35.3%) of 34 patients with germline TP53 variants did not meet LFS testing criteria. Loss of heterozygosity of germline TP53 variant was observed in 96.0% (95% confidence interval [CI] = 79.7% to 99.9%) of core LFS spectrum-type tumors vs 45.5% (95% CI = 16.8% to 76.6%) of other tumors and 91.3% (95% CI = 72.0% to 98.9%) of tumors from patients who met LFS testing criteria vs 61.5% (95% CI = 31.6% to 86.1%) of tumors from patients who did not. Tumors retaining the wild-type TP53 allele exhibited wild-type p53 expression. Our results indicate that some TP53 variants identified in blood-only sequencing are not germline and a substantial proportion of patients with LFS are missed based on current testing guidelines. Additionally, a subset of tumors from patients with LFS do not have biallelic TP53 inactivation and may represent cancers unrelated to their germline TP53 defect.

Pathogenic constitutional APC variants underlie familial adenomatous polyposis, the most common hereditary gastrointestinal polyposis syndrome. To improve variant classification and resolve the interpretative challenges of variants of uncertain significance (VUSs), APC-specific variant classification criteria were developed by the ClinGen-InSiGHT Hereditary Colorectal Cancer/Polyposis Variant Curation Expert Panel (VCEP) based on the criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP). A streamlined algorithm using the APC-specific criteria was developed and applied to assess all APC variants in ClinVar and the International Society for Gastrointestinal Hereditary Tumours (InSiGHT) international reference APC Leiden Open Variation Database (LOVD) variant database, which included a total of 10,228 unique APC variants. Among the ClinVar and LOVD variants with an initial classification of (likely) benign or (likely) pathogenic, 94% and 96% remained in their original categories, respectively. Incontrast, 41% ClinVar and 61% LOVD VUSs were reclassified into clinically meaningful classes, the vast majority as (likely) benign.The total number of VUSs was reduced by 37%. In 24 out of 37 (65%) promising APC variants that remained VUS despite evidence for pathogenicity, a data-mining-driven work-up allowed their reclassification as (likely) pathogenic. These results demonstrated that the application of APC-specific criteria substantially reduced the number of VUSs in ClinVar and LOVD. The study also demonstrated the feasibility of a systematic approach to variant classification in large datasets, which might serve as a generalizable model for other gene- or disease-specific variant interpretation initiatives. It also allowed for the prioritization of VUSs that will benefit from in-depth evidence collection. This subset of APC variants was approved by the VCEP and made publicly available through ClinVar and LOVD for widespread clinical use.