Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome associated with a highly penetrant cancer spectrum characterized by germline TP53 mutations. We characterized the first LFS zebrafish hotspot mutants, tp53 R217H and R242H (human R248H and R273H), and found these mutants exhibit partial-to-no activation of p53 target genes, have defective cell-cycle checkpoints, and display partial-to-full resistance to apoptosis, although the R217H mutation has hypomorphic characteristics. Spontaneous tumor development histologically resembling human sarcomas was observed as early as 6 months. tp53 R242H mutants had a higher lifetime tumor incidence compared to tp53 null and R217H mutants, suggesting it is a more aggressive mutation. We observed mutation-specific tumor phenotypes across tp53 mutants with associated diverse transcriptomic and DNA methylome profiles in tp53 mutant larvae, impacting metabolism, cell signalling, and biomacromolecule synthesis and degradation. These tp53 zebrafish mutants demonstrate fidelity to their human counterparts and provide new insights into underlying tumorigenesis mechanisms and kinetics that suggest metabolic rewiring and cellular signalling changes occur prior to tumor initiation, which will guide targeted therapeutics for LFS.

Germline pathogenic TP53 variants predispose individuals to a high lifetime risk of developing multiple cancers and are the hallmark feature of Li-Fraumeni syndrome (LFS). Our group has previously shown that LFS patients harbor shorter plasma cell-free DNA fragmentation; independent of cancer status. To understand the functional underpinning of cfDNA fragmentation in LFS, we conducted a fragmentomic analysis of 199 cfDNA samples from 82 TP53 mutation carriers and 30 healthy TP53-wildtype controls. We find that LFS individuals exhibit an increased prevalence of A/T nucleotides at fragment ends, dysregulated nucleosome positioning at p53 binding sites, and loci-specific changes in chromatin accessibility at development-associated transcription factor binding sites and at cancer-associated open chromatin regions. Machine learning classification resulted in robust differentiation between TP53 mutant versus wildtype cfDNA samples (AUC-ROC = 0.710–1.000) and intra-patient longitudinal analysis of ctDNA fragmentation signal enabled early cancer detection. These results suggest that cfDNA fragmentation may be a useful diagnostic tool in LFS patients and provides an important baseline for cancer early detection. Here, Wong et al investigate the cell-free DNA landscape of individuals with Li-Fraumeni syndrome (LFS), a cancer predisposition, and find altered composition compared to non-LFS individuals which can be used to detect and track cancer development.

ABSTRACT Li-Fraumeni syndrome (LFS) has recently been redefined as a ‘spectrum’ cancer predisposition disorder to reflect its broad phenotypic heterogeneity. The wide functional gradient associated with different TP53 variants is thought to contribute to LFS heterogeneity, although it is still poorly understood and there is an unmet clinical need for risk stratification strategies. Leveraging p53 mutagenesis dataset, we performed an unsupervised cluster analysis that revealed five TP53 variant clusters with unique structural and functional consequences. Classifying variant carriers according to these clusters stratified cancer onset and survival using discovery and validation cohorts, and exposed important clinical characteristics to consider for patient management. In particular, we identified a subgroup of monomeric TP53 variant carriers prone to osteosarcoma, along with a cluster associated with less “LFS-like” phenotypes enriched in carriers with no history of cancer. Our classification of TP53 variants demonstrates the existence of a wide TP53- heritable cancer susceptibility spectrum and provides a new framework to delineate carriers toward personalized patient care.

Abstract Li-Fraumeni syndrome (LFS) is a highly penetrant cancer predisposition disorder caused by germline variants in the TP53 tumor suppressor gene. LFS has recently been redefined as a ‘spectrum’ disorder to reflect the highly variable cancer types with largely unpredictable ages-of-onset and disease severity. The broad functional gradient associated with different TP53 variants is thought to contribute to LFS heterogeneity, although it is still poorly understood and there is an unmet clinical need for risk stratification strategies to improve variant interpretation and patient care. Here, we performed an unsupervised cluster analysis leveraging p53 mutagenesis datasets (Kato et al., 2003 & Giacomelli et al., 2018) that revealed five TP53 mutational clusters with unique mutation patterns, structural features, and functional consequences. Stratifying germline carriers based on the five clusters exposed important clinical characteristics to consider for patient management, such as ages-of-onset, tumor type development, and survival outcomes. In particular, we discovered an osteosarcoma-prone subgroup comprised of monomeric mutant p53 carriers with aggressive cancer phenotypes. We also identified a cluster of TP53 variants found more often in non-cancer and healthy older populations, and carriers of these variants that developed cancer had less “LFS-like” phenotypes including an older age-of-onset and a significantly higher frequency of colorectal cancers. Remarkably, our TP53 variant clustering strategy could also stratify breast cancer survival among germline carriers. This work provides a new framework to delineate the LFS spectrum toward the development of machine learning-based approaches for personalized cancer surveillance plans. Citation Format: Nicholas W. Fischer, Brianne Laverty, Ran Kafri, Kara N. Maxwell, Emma R. Woodward, Christian Kratz, David Malkin. TP53 mutational clusters stratify the Li-Fraumeni syndrome spectrum [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6198.

Abstract Background: Transposable elements (TEs) are dynamic repetitive regions which generate mutations and structural variants. TP53 plays a crucial role in suppressing TE movement to maintain genomic stability. The relationship between TP53 and TEs has been extensively studied in tumours, but not the germline. Individuals with germline TP53 pathogenic variants have Li-Fraumeni Syndrome (LFS), a cancer predisposition syndrome with a high lifetime risk of cancer in various tissues. This study aims to characterize the TE landscape in individuals with Li-Fraumeni Syndrome and determine how this contributes to their increased cancer risk. Methods: MELT and xTea were used to identify TEs in children with (n=48) or without (n=198) a germline TP53 variant. TE calls were merged with SURVIVOR and variants were annotated with AnnotSV. To assess the influence of TP53 on TE location, we quantified TEs across genomic windows and identified the top 100 significantly different regions with the Mann-Whitney U test, adjusting for multiple comparisons. We used these regions to develop a gradient-boosted tree model with 5-fold cross-validation to predict TP53 status. Results: Individuals with germline TP53 variants harboured significantly fewer ALU and LINE1 elements in their germline genome compared to the control dataset (p<0.001). The phenomenon was consistent across chromosomes and significant in chromosomes 5 and 11 (FDR<0.05). A gradient-boosted tree model was able to differentiate patients with and without a germline TP53 variant with an AUPRC of 0.77 on an unseen test set. Conclusion: Germline TP53 variants may effect the frequency and location of germline TE insertions, which may influence nearby genomic variations and lead to cancer development. Analyzing TEs in individuals with LFS will enhance our understanding of accelerated cancer development in these patients, informing future research for diagnostic and therapeutic approaches. Citation Format: Brianne Laverty, Shilpa Yadahalli, Vallijah Subasri, David Malkin. The role of TP53 on transposable elements in pediatric cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A010.

Abstract Li-Fraumeni syndrome (LFS) has recently been redefined as a ‘spectrum’ cancer predisposition disorder to reflect its broad phenotypic heterogeneity. The wide functional gradient associated with different TP53 variants is thought to contribute to LFS heterogeneity, although it is still poorly understood and there is an unmet clinical need for risk stratification strategies. Leveraging p53 mutagenesis datasets, we performed an unsupervised cluster analysis that revealed five TP53 variant clusters with unique structural and functional consequences. Classifying variant carriers according to these clusters stratified cancer onset and survival using discovery and validation cohorts, and exposed important clinical characteristics to consider for patient management. In particular, we identified a subgroup of monomeric TP53 variant carriers prone to osteosarcoma, along with a cluster associated with less “LFS-like” phenotypes enriched in carriers with no history of cancer. Our classification of TP53 variants demonstrates the existence of a wide TP53-heritable cancer susceptibility spectrum and provides a new framework to delineate carriers toward personalized patient care. Citation Format: Nicholas W. Fischer, Brianne Laverty, Noa Alon, Emilie Montellier, Pierre Hainaut, Kara N. Maxwell, Christian P. Kratz, David Malkin. TP53 variant clusters stratify the Li-Fraumeni spectrum and reveal an osteosarcoma-prone subgroup [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A005.

Purpose: An expected outcome following germline genome sequencing in oncology is the discovery of ‘secondary findings’ (SFs). SFs comprise pathogenic(P)/likely P (LP) germline variants in cancer genes not typically associated with the presenting cancer, in addition to germline variants of uncertain significance (VUS) to the patient’s cancer. Due to the rarity of childhood cancers and a dearth of studies analyzing SFs, many pediatric SFs are categorized as VUS without clinical interpretation. Interpreting SFs poses significant challenges: VUSs and other SFs are frequently not included in clinical molecular reports, and even when reported (often through research), their clinical utility and long-term impact on patient health are unclear. However, we know VUSs can have clinical importance because some VUSs, when investigated thoroughly, have been reclassified as pathogenic predictors of significant health conditions in children. We hypothesize that an in-depth characterization of the landscape of germline SFs/VUSs across a diverse pediatric cancer cohort will reveal new roles of these genes and mutations in pediatric cancers. Methods: To explore germline SFs in pediatric cancer patients, we analyzed germline whole-genome sequencing (WGS) data for patients with rare, relapsed, refractory, and metastatic childhood cancers enrolled in the SickKids Cancer Sequencing Program (KiCS). We developed a custom analysis pipeline to identify germline single-nucleotide variants and indels deemed SFs, auto-classify their pathogenicity (ex. P, LP, or VUS) using CharGer, filter for PanCanAtlas-indicated cancer predisposition genes, and sort the remaining variants by cancer and non-cancer associations. Results: The KiCS cohort (n = 511) encompassed over 133 different tumor types; the median age of participants was 14 years (SD = 10.27) and 55% of patients were male. Ongoing work in our lab will catalogue the frequency and distribution of SFs in KiCS and analyze germline variants by subgroup (gene, tumor subtype, stage, demographics, gene function). We will also compare SF prevalence in KiCS to the general population using the gnomAD dataset. Results from preliminary analyses of this cohort will be presented. Significance: SFs/VUSs are under-utilized in cancer management. This work advances the holistic understanding of germline genomics in pediatric oncology and the roles of SFs in disease. Future studies will evaluate SFs by patient ancestry and validate cancer associations through the evaluation of allelic imbalance/loss of heterozygosity in matched tumor genomes. Citation Format: Safa Majeed, Stephenie Prokopec, Brianne Laverty, Vallijah Subasri, Michael Taylor, Yvonne Bombard, Trevor Pugh, Adam Shlien, Anita Villani, David Malkin. Investigating secondary findings in a pediatric cancer cohort: preliminary findings. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6545.

Background: Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome caused by germline mutations in the tumor suppressor gene TP53. LFS is estimated to occur in 1:1000 - 1:5000 people and is associated with a 80% lifetime cancer risk. This syndrome is diagnosed using familial cancer history and germline TP53 sequencing; however, clinical heterogeneity and variants of unknown significance limit diagnostic precision. Accurate diagnosis is imperative to implement surveillance for secondary malignancies and familial genetic testing. Methods: We hypothesized that LFS cancers evolve uniquely from sporadic cancers, implying that the somatic genomes of LFS patients exhibit distinct characteristics that can infer the predisposition syndrome. To investigate this, we interrogated mutational signatures, TP53 copy number, TP53 loss of heterozygosity, ploidy, and the incidence of chromothripsis in tumor compared to normal samples (blood or fibroblast) of individuals with germline TP53 mutations (n=27), somatic TP53 mutations (n=17) and WT for TP53 (n=158). We created a random forest model with 10-fold cross validation to determine if somatic features could diagnose LFS. Results: No signatures were significantly associated with LFS (Wilcoxon rank-sum test, Benjamin-Hochberg FDR correction). As previously reported in the literature, LFS compared to non-LFS cancers in our cohort were more likely to: be hyperdiploid (odds ratio (OR) = 11.83, FDR < 0.0001, Fisher exact test), have undergone TP53 loss of heterozygosity (OR = 23.15, FDR < 0.0001, Fisher exact test) and experience chromothripsis (OR = 7.76, FDR < 0.001, Fisher exact test). The area under the receiver operating curve (AUROC) for our random forest model with 10-fold cross validation was 0.90, the area under the precision recall curve (AUPRC) was 0.59, the positive predictive value (PPV) was 0.70, the negative predictive value (NPV) was 0.93 and the F1-score was 0.52. This implies that the somatic genomic features are reliable indicators of this germline syndrome. We have obtained access to a future 50 LFS samples from the Pediatric Cancer Genome Project dataset, which we hope will improve our model’s performance. Conclusion: We have developed a machine learning tool that uses somatic features to identify LFS, a germline cancer predisposition syndrome. As the importance of precision oncology becomes apparent, a tool to identify LFS patients from the somatic genome will facilitate early diagnosis. This will allow individuals to enter a surveillance program for early detection of secondary tumors, leading to improved outcomes. Citation Format: Brianne Laverty, Vallijah Subasri, Nicholas Light, David Malkin. Diagnosing Li-Fraumeni syndrome from the somatic genome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3364.