Gaëlle Bougeard

Introduction:Next-generation sequencing performed for therapeutic decision-making in pediatric cancers provides an opportunity to identify germline variants associated with hereditary cancer predisposition and other actionable inherited conditions. Methods:Germline whole-exome sequencing (WES) was performed within the MAPPYACTS (MoleculAr Profiling for Pediatric and Young Adult Cancer Treatment Stratification) trial. Germline data were available for 674 patients with diverse relapsed or refractory malignancies. Families provided informed consent regarding disclosure of actionable germline findings. A multidisciplinary committee established a panel of 184 cancer predisposition genes with established surveillance recommendations and 49 medically actionable noncancer genes from the ACMG SF v3.1 list. Variants were analyzed using bioinformatic pipelines including quality filtering, ClinVar annotation, and computational pathogenicity assessment. Results:Overall, 98.6% of families consented to the disclosure of actionable genetic findings. Among 674 patients, 130 likely pathogenic or pathogenic variants (LPV/PV) were identified in 130 patients (19.3%) across 53 cancer predisposition genes. Based on inheritance patterns and clinical relevance, genetic counseling was recommended for 58 patients (8.6%). The most frequently altered genes were TP53 (n=17), DICER1 (n=5), and BRCA1 (n=4). Predisposition findings corresponded to the expected tumor spectrum in 48% of cases, and 35% had not been previously recognized. Additionally, 10 patients (1.5%) carried actionable LPV/PV in noncancer genes associated with cardiomyopathies, familial hypercholesterolemia, or transthyretin amyloidosis. Discussion:Systematic germline WES integrated into theranostic profiling may provide additional clinical value through the identification of actionable cancer predispositions and other inherited conditions. We propose recommendations for a structured, multidisciplinary framework to support clinical decision-making.

Consensus classification of hereditary hematologic malignancies

Abnormal RNA splicing is an underrecognized driver of pathogenicity in germline TP53 — the cause of Li–Fraumeni syndrome (LFS). We re-evaluated exonic single-nucleotide variants (SNVs) that yield missense or synonymous changes for spliceogenic effects by integrating SpliceAI prediction, in-vitro minigene assays, and analysis of tumor RNA-seq from TCGA, and assessed genotype-phenotype correlations using clinical data from multiple databases and national registries. We identified 58 spliceogenic exonic SNVs (SE-SNVs) across the TP53 gene (40 missense, 18 synonymous). Experimental validation confirmed aberrant splicing for 15 out of 17 tested variants, most often through cryptic splice-site activation that introduced frameshifts and premature termination. Clinically, carriers of SE-SNVs previously considered as mild or of low-pathogenicity by protein-based assays showed earlier onset and LFS-signature cancers, indicating that splicing disruption can override amino-acid effects. The recurrent c.375 G > A (p.(Thr125 = )) showed heterogeneous effect: with both childhood/adolescent and adult onset, consistent with partial, variable retention of canonical splicing. These data reveal a substantial burden of spliceogenic pathogenicity in TP53 and strong support integrating splicing prediction, functional validation, and transcript-level evidence into variant interpretation and risk stratification in LFS.

Pathogenic germline variants in the TP53 gene cause Li-Fraumeni syndrome (LFS), a highly penetrant cancer predisposition disorder. Most of these variants arise from single-nucleotide variations (SNVs) in TP53 exons, causing missense mutations. However, some of these SNVs may also alter mRNA splicing, defining spliceogenic single nucleotide variants (SE-SNVs) of uncertain clinical significance. We reassessed previously classified TP53 missense variants for spliceogenic effects using SpliceAI predictions, in vitro minigene assays, and transcriptomic data from TCGA. Genotype-phenotype correlations were evaluated using clinical data from carriers of TP53 germline variants across multiple databases and registries. Among 58 identified SE-SNVs, 40 were missense and 18 synonymous. Experimental validation showed that most induce aberrant splicing events, frequently via cryptic splice site activation, leading to frameshift and premature stop codons. Several missense variants previously classified as having mild or low pathogenicity were found instead to have strong spliceogenic effects and were associated with early-onset cancers typical of LFS, suggesting that splicing alterations may override their protein-coding impact. The frequent SNV c.375G>A leading to the synonymous variant p.T125= shows intermediate severity, likely due to partial retention of normal splicing activity. Our study highlights the underestimated pathogenic potential of SE-SNVs affecting the TP53 gene. These findings underscore the importance of integrating splicing predictions, functional assays, and transcript-level analyses into TP53 variant interpretation to improve risk stratification in LFS.

STRUCTURED Importance: Li-Fraumeni Syndrome (LFS) is an heterogenous cancer predisposition caused by pathogenic TP53 variants, characterized by a lifelong high risk of a broad spectrum of cancers. At least certain pathogenic TP53 variants have been shown be immunogenic in a somatic context. Whether neoantigenicity contributes to the heterogeneity of LFS is unknown. Objective: To analyze the correlations between predicted neoantigenic properties of pathogenic TP53 missense variants and LFS patterns. Design: Association study between predicted MHC-I presentation scores for TP53 hotspot variants, LFS presentation and individual HLA-I genotyping in carriers of TP53 germline pathogenic variants using data from mutation databases and clinical registries. Setting: MHC-I presentation scores were generated for the set of nonameric neo-peptides surrounding each TP53 missense variant against 145 different HLA-I using NetMHCpan 4.1 and the Allele Frequency Net Database. Genotype-phenotype data were leveraged from the public TP53 database (germline dataset, n=3,446; https://tp53.isb-cgc.org/ ) and two independent LFS clinical registries (n=339). Individual correlations between HLA-I genotyping, TP53 missense variants and phenotypes were investigated in a group of 173 subjects with LFS. Participants: Individuals with LFS enrolled in LFS-registries in France and Germany were included. Main outcomes and measures: A predicted neoantigenic score (PNS) was calculated for each variant. Correlations with median age at first cancer, and cancer type were analyzed. Results: Among 709 carriers of frequent TP53 pathogenic variants, PNS was strongly correlated with median age at first cancer (range 18-43 years, p=0.01319, R=0.69). Compared to carriers of low PNS (2) variants showed delayed median age at first diagnosis (34 [CI95%, 29-40] vs. 25 years [CI95%, 22-27]), fewer sarcomas (osteosarcoma [RR 0.29, p-adj = 0.02]; soft-tissue [RR 0.41, p-adj = 0.02]), and more cancer types typically not associated with LFS [RR 1.61, p-adj = 0.02]. Conclusion and Relevance: MHC-I neoantigenic properties of TP53 variants modify cancer risk and spectrum in carriers of pathogenic TP53 variants, suggesting that individual variant-specific immune response may contribute to the heterogenous presentation of LFS.

Li–Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition condition characterized by a high lifetime risk for a wide spectrum of malignancies associated with germline pathogenic/likely pathogenic variants in the TP53 tumor suppressor gene. Secondary malignant neoplasms are particularly common. Early cancer detection through surveillance enables early intervention and leads to improved clinical outcomes with reduced tumor-related mortality and treatment-related morbidity. Since the 2017 publication of LFS tumor surveillance guidelines from the inaugural American Association for Cancer Research Childhood Cancer Predisposition Workshop, understanding the genotype–phenotype relationships in LFS has evolved, and adaptations of the guidelines have been implemented in institutions worldwide. The “Toronto Protocol” remains the current standard for lifelong surveillance; however, as outlined in this perspective, modifications should be considered about the use of certain modalities to target organs in an age-dependent manner. The Working Group’s recommendations have also been extended to include a more detailed outline for surveillance in the adult TP53 pathogenic/likely pathogenic variant carrier population, based on the recognition that early education of both practitioners and patients on what to expect after the transition from childhood/adolescence to young adulthood is important in preparing them for changes in surveillance strategies. In this perspective, we provide an up-to-date clinical overview of LFS and present our updated consensus tumor surveillance recommendations from the 2023 American Association for Cancer Research Childhood Cancer Predisposition Workshop.

Background: Li-Fraumeni syndrome (LFS) predisposes individuals to a wide range of cancers from childhood onwards, underscoring the crucial need for accurate interpretation of germlineTP53variants for optimal clinical management of patients and families. Several unclassified variants, particularly those potentially affecting splicing, require specialised testing. One such example is the NM_000546.6:c.1101-2A>C (rs587781664) variant, located at the splice acceptor site of the last intron ofTP53, identified in a female patient with breast cancer diagnosed in her 20s.Methods: To interpret this variant, which has been classified as a variant of uncertain significance (VUS), we developed specific assays including a p53 functional assay, RT-QMPSF, Splice and Expression Analyses by exon Ligation and High-Throughput Sequencing and long RT-droplet digital PCR.Results: We demonstrated a loss of p53 transcriptional activity, and a half reduction in TP53 mRNA expression. Additionally, we detected the use of a novel alternative last exon downstream of exon 11, which we have named exon 12. This transcript, typically detectable at low levels in most individuals, was found to be more highly expressed in the c.1101-2A>C carrier, predominantly transcribed from the mutant allele due to the disruption of the splice acceptor site in intron 10.Conclusion: By combining these approaches, we successfully reclassified this intronic VUS as ‘pathogenic’, enabling appropriate genetic counselling for the patient and her family. Additionally, we identified a novel TP53 alternative transcript that is expressed in both physiological and pathological contexts, with heightened expression in the patient with LFS. This discovery provides a basis for further investigation into the role of TP53 isoforms in LFS oncogenesis.