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Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer-predisposition disorder. Approximately 70% of individuals who fit the clinical definition of LFS harbor a pathogenic germline variant in the TP53 tumor suppressor gene. However, the remaining 30% of patients lack a TP53 variant and even among variant TP53 carriers, approximately 20% remain cancer-free. Understanding the variable cancer penetrance and phenotypic variability in LFS is critical to developing rational approaches to accurate, early tumor detection and risk-reduction strategies. We leveraged family-based whole-genome sequencing and DNA methylation to evaluate the germline genomes of a large, multi-institutional cohort of patients with LFS (n = 396) with variant (n = 374) or wildtype TP53 (n = 22). We identified alternative cancer-associated genetic aberrations in 8/14 wildtype TP53 carriers who developed cancer. Among variant TP53 carriers, 19/49 who developed cancer harbored a pathogenic variant in another cancer gene. Modifier variants in the WNT signaling pathway were associated with decreased cancer incidence. Furthermore, we leveraged the noncoding genome and methylome to identify inherited epimutations in genes including ASXL1, ETV6, and LEF1 that confer increased cancer risk. Using these epimutations, we built a machine learning model that can predict cancer risk in patients with LFS with an area under the receiver operator characteristic curve (AUROC) of 0.725 (0.633–0.810). Significance: Our study clarifies the genomic basis for the phenotypic variability in LFS and highlights the immense benefits of expanding genetic and epigenetic testing of patients with LFS beyond TP53. More broadly, it necessitates the dissociation of hereditary cancer syndromes as single gene disorders and emphasizes the importance of understanding these diseases in a holistic manner as opposed to through the lens of a single gene.

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li–Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. Significance: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027

Based on current studies, the incidence of Ewing sarcoma (ES) varies significantly by race and ethnicity, with the disease being most common in patients of European ancestry. However, race/ethnicity has generally been self-reported rather than formally evaluated at a population level using DNA evidence. Additionally, mitochondrial dysfunction is a hallmark of ES, yet there have been no reported studies of mitochondrial genetics in ES. Thus, we evaluated both the mitochondrial and nuclear ancestries of 420 pediatric ES patients in the United States using whole-genome sequencing. We found that the mitochondrial DNA (mtDNA) genomes of only six (1.4 %) patients belonged to African L haplogroups, while those of 90 % of the patients belonged to macrohaplogroup R, which includes haplogroup H, the most common maternal lineage in Europe. Compared to the general US population, European haplogroups were significantly enriched in ES patients (p 2.2e-16) and the African haplogroups are significantly impoverished (p 4.6e-16). Using the ancestry informative markers defined in a National Genographic study, the vast majority of patients exhibited significant nuclear ancestry originating from the Mediterranean, Northern Europe, and Southwest Asia, including all six patients with African L mtDNAs. Very few had primarily African nuclear ancestry. This is the first genomic epidemiology study to simultaneously interrogate the mitochondrial and nuclear ancestries of ES patients. While supporting previous findings of enriched European ancestry in ES patients, these results also suggest alternative hypotheses for the significant contribution of mitochondrial ancestry in ES patients, as well as the protective role of African ancestry.

Germline pathogenic variants in cancer susceptibility genes are identified in up to 18% of all children with cancer. Because pediatric cancer predisposition syndromes (CPS) themselves are rare and underrecognized, there are limited data to guide the diagnosis and management of affected children and at-risk relatives. Furthermore, the care of affected children requires distinct considerations given the early onset of cancers, lifelong risks of additional cancers, and potential late effects of therapy. Herein, we discuss efforts to leverage existing infrastructure, organize experts, and develop a new consortium to optimize care and advance research for children with CPS. A 2016 workshop organized by the American Association for Cancer Research united many experts in childhood cancer predisposition and resulted in publication of multiple consensus guidelines for tumor surveillance. More recently, several of these authors established the Consortium for Childhood Cancer Predisposition (C3P), a multi-institutional collaboration that provides a structure for systematic research in cancer predisposition, screening, and prevention in children. The Consortium intends to work with other cooperative groups to merge longitudinal data from children with CPS throughout the continuum of the cancer risk period, as well as cancer treatment and survivorship care, to optimize overall outcomes.

Supplementary Figures 1-9 demonstrating the GOF and LOF activities of p53(A347D)

Differentially expressed genes in U2OS cells varying in p53 allelic status combined from two independent RNA library preparations

p53 ChIPseq peaks from U2OS cells varying in p53 allelic status

p53 ChIPseq peaks from osteoblasts derived from normal and LFS individuals

Secondary constitutional epimutations in LFS. A, Concordance between enrichment scores of 931 EWAS probes found significantly associated with cancer status in the discovery cohort and the validation cohort, for both the internal (left) and external (right) validation cohort. Blue = associated with cancer status and a cis-CSCE; Red = associated with cancer status but not a cis-CSCE. B, Manhattan plot of adjusted P values from meQTL association tests between the 931 EWAS probes and their most correlated rsSNP. Red line: FDR = 0.1; Blue line: FDR = 0.01; Green points: probes in cancer genes with FDR < 0.1. C, Top cis-CSCE in ETV6, TET3, MIR143, ASXL1, PARVB, and LRG1.D, Feature enrichment heatmap of the 259 significant cis-CSCE loci showing adjusted P value (p) and effect size (d). E, ROC curve of the epimutation cancer risk model on the test set. F, UMAP projection of methylation at 259 cis-CSCE for all patients, colored by cancer status. G, UMAP projection of methylation at 259 cis-CSCE for all patients, colored by clusters associated with risk. H, Kaplan–Meier survival curve demonstrating significant differences in survival probability between the three epimutation clusters.

Variants in cancer predisposition genes (class 1–2) in the wildtype and variant TP53 cohorts, annotated with cancer type, sex, LFS criteria, inheritance status, segregation, variant location, variant function, and zygosity

cisCSCE

Pedigrees and class 1–3 variants for two wildtype TP53 families that fit the classic LFS criteria. A, Pedigree of wildtype TP53 family (WT4) that fits the classic LFS criteria with four family members sequenced, two that developed cancer and two that are cancer-free. B, Pedigree of wildtype TP53 family (WT6) that fits the classic LFS criteria with six family members sequenced: five that developed cancer and one that is cancer-free.

Genomics informed risk stratification guidelines for patients with LFS with a germline TP53 variant

Tier 1-3 Gene Lists

Supplementary Figures

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer-predisposition disorder. Approximately 70% of individuals who fit the clinical definition of LFS harbor a pathogenic germline variant in the TP53 tumor suppressor gene. However, the remaining 30% of patients lack a TP53 variant and even among variant TP53 carriers, approximately 20% remain cancer-free. Understanding the variable cancer penetrance and phenotypic variability in LFS is critical to developing rational approaches to accurate, early tumor detection and risk-reduction strategies. We leveraged family-based whole-genome sequencing and DNA methylation to evaluate the germline genomes of a large, multi-institutional cohort of patients with LFS (n = 396) with variant (n = 374) or wildtype TP53 (n = 22). We identified alternative cancer-associated genetic aberrations in 8/14 wildtype TP53 carriers who developed cancer. Among variant TP53 carriers, 19/49 who developed cancer harbored a pathogenic variant in another cancer gene. Modifier variants in the WNT signaling pathway were associated with decreased cancer incidence. Furthermore, we leveraged the noncoding genome and methylome to identify inherited epimutations in genes including ASXL1, ETV6, and LEF1 that confer increased cancer risk. Using these epimutations, we built a machine learning model that can predict cancer risk in patients with LFS with an area under the receiver operator characteristic curve (AUROC) of 0.725 (0.633–0.810).Significance:Our study clarifies the genomic basis for the phenotypic variability in LFS and highlights the immense benefits of expanding genetic and epigenetic testing of patients with LFS beyond TP53. More broadly, it necessitates the dissociation of hereditary cancer syndromes as single gene disorders and emphasizes the importance of understanding these diseases in a holistic manner as opposed to through the lens of a single gene.

Germline genetic variants in LFS. A, Classification scheme for genetic variants identified from WGS. The pyramid (top) shows how genes were prioritized into four tiers from highest known cancer predisposition potential to lowest. All variants were then classified using a Cancer Variant Classification Schema (Materials and Methods) into five classes (bottom) based on pathogenicity and whether the gene fell into Tier 1–4. AD: autosomal dominant; AR: autosomal recessive; CPG: cancer predisposition gene. B, Frequency of P/LP variants in known cancer genes (class 1–3) in LFS, KiCS and 1kGP. Fisher exact test P values for comparisons between the LFS cohorts and KiCS/1kGP shown. C, Kaplan–Meier survival curve comparing patients with P/LP variants in the WNT signaling pathway (yellow), patients with at least one class 1–3 variant (red) and patients with neither WNT signaling or class 1–3 variants (blue). D, Landscape of genetic alterations in cancer genes (class 1–3) in LFS. Annotated with family, cancer type and TP53 variant status. Genetic variants are indicated as a deletion (blue), SNV or indel (red) or duplication (purple). E, The proportion of individuals in the wildtype and variant TP53 cohorts with classes 1–5 variants (Materials and Methods). F, The number of P/LP variants in cancer genes (class 1–3) in individuals that developed cancer compared with unaffected individuals, stratified by TP53 status. G, The number of P/LP variants (class 1–3) in wildtype versus variant TP53 carriers, stratified by cancer status. On the basis of the premise that variants in the WNT signaling pathway are associated with decreased cancer incidence in LFS, these variants were removed from the comparisons made in F and G.

LFS Cohort

1000G Variants

LFS Variants

Differential regulation of WNT signaling and LEF1 in LFS. A, Probes in the WNT signaling pathway (WNT4, SMAD3, SFRP4, SERPINF) significantly associated with cancer status. B, Probe in the body of LEF1 (cg03041109) associated with cancer status (FDR = 0.07). C, Methylation of probe in body of LEF1 (cg03041109) across members within each LFS family. Probands almost unanimously have higher LEF1 methylation than their unaffected family members. D, Plasma cfDNA methylation of the body of LEF1 (chr4:109056901-109057200), between patients with LFS and HBCs. E, Tumor methylation of the body of LEF1 (cg03041109) in CPTs, with and without a germline variant in TP53.

The LFS cohort. A, Patient cohort and characteristics (age of first cancer onset, tumor type) by TP53 status and whether profiling was performed using WGS, methylation or both. AA = adrenal adenoma; ACC = adrenocortical carcinoma; ALL = acute lymphoid leukemia; CPC = choroid plexus carcinoma; CSA = chondrosarcoma; GCT = germ cell tumor; LMS = leiomyosarcoma; MFH = malignant fibrous histiocytoma; MFS = myxofibrosarcoma; NHL = non–Hodgkin lymphoma; OS = osteosarcoma; RMS = rhabdomyosarcoma. B, Lollipop plot displaying the location and number of SNVs and indels on the canonical TP53 transcript: NM_000546, which consists of a TAD, DBD, and tetramerization domain. TP53 hotspot variants present in our cohort (175, 245, 248, 273, 282) are highlighted.

Characteristics of LFS patients' fibroblasts and derivatives

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li–Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein.Significance:A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches.See related commentary by Stieg et al., p. 1046.See related article by Gencel-Augusto et al., p. 1230.This article is highlighted in the In This Issue feature, p. 1027

Differentially expressed genes in U2OS cells varying in p53 allelic status

p53 ChIPseq peaks from U2OS cells varying in p53 allelic status

Summary of GOF vs. LOF activities of p53(A347D) in U2OS cells

Histone deacetylase (HDAC) inhibition has sporadic clinical efficacy in urothelial carcinoma; the genomic basis for clinical response is not known. In two separate phase I clinical trials testing pharmacokinetic aspects of HDAC inhibitors in advanced solid tumors, we identified one patient with advanced urothelial carcinoma who had a complete response to belinostat, and one patient with advanced urothelial carcinoma who had a partial response to panobinostat. The archived tumors of the responders were genomically characterized in comparison to others with urothelial carcinoma on the trials. Urothelial carcinoma cell lines treated with panobinostat and belinostat were studied to elucidate the mechanisms of benefit. Notably, the urothelial carcinoma tumors that responded to HDAC inhibition had ARID1A mutations. ARID1A mutations were also noted in the tumors of three patients who had stable disease as their best response to HDAC inhibition. Corroborating the basis of sensitivity, transcriptional profiling of platinum-resistant ARID1A-mutated HT1197 cells treated with panobinostat reveals negative enrichment for both cyto-proliferative (MYC and E2F targets) and DNA repair gene sets, and positive enrichment for TP53 and inflammatory gene sets. Our study identifies ARID1A loss as a basis for clinical response to pan HDAC inhibition and offers avenues for potential rational therapeutic combinations with HDAC inhibitors in advanced urothelial carcinoma.

GSEA with Hallmark gene sets performed on transcriptional changes in HT1197 cells treated with panobinostat for 48 hours. A. Top 6 downregulated GSEA signatures B. Top 6 upregulated GSEA signatures C. GSEA HT1197 vs UMUC3 ( comparison of transcriptome of untreated samples reveals significantly greater presence of the E2F target gene signature in HT1197 cells).

Supplementary Figure 1 - PDF file 1233K, Supplemental Figure 1: Representative array comparative genomic hybridization (aCGH) results of genomic copy number near-normal GIST (left column, n=5) and genomic copy number abnormal GIST (right column, n=5) (Agilent 180K catalogue assay)

Heat map of top 50 genes affected in HT1197 cells when treated with panobinostat at 48 hours log2 gene expression change(HT1197 A-B and C represent control replicates, HT1197 Pano A, B, C represent treated replicates). For the cell lines with replicates, statistically significant genes were identified with a FDR of 2 fold. Plot was generated with R using median-centered regularized log2 values.

LFS Cohort

Supplementary Figures

KiCS Variants

Individual drug characteristics and doses, maximum plasma levels and protein binding of panobinostat and belinostat in the two phase 1 studies

Tier 1-3 Gene Lists

cisCSCE

Study Schema

Supplementary Table 1 XLS file 22K, Tumors and reference tissues for methylation microarray analysis

Swimmer Plot to show the best overall response and duration of benefit in seven bladder cancer patients from Table 1 treated with HDAC inhibition.

Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer-predisposition disorder. Approximately 70% of individuals who fit the clinical definition of LFS harbour a pathogenic germline variant in the TP53 tumour suppressor gene. However, the remaining 30% of patients lack a TP53 variant and even among variant TP53 carriers, approximately 20% remain cancer-free. Understanding the variable cancer penetrance and phenotypic variability in LFS is critical to developing rational approaches to accurate, early tumour detection and risk-reduction strategies. We leveraged family-based whole-genome sequencing and DNA methylation to evaluate the germline genomes of a large, multi-institutional cohort of LFS patients (n=396) with variant (n=374) or wildtype TP53 (n=22). We identified alternative cancer-associated genetic aberrations in 8/14 wildtype TP53 carriers who developed cancer. Among variant TP53 carriers, 19/49 who developed cancer harboured a pathogenic variant in another cancer gene. Modifier variants in the WNT signaling pathway were associated with decreased cancer incidence. Furthermore, we leveraged the non-coding genome and methylome to identify inherited epimutations in genes including ASXL1, ETV6 and LEF1 that confer increased cancer risk. Using these epimutations, we built a machine learning model that can predict cancer risk in LFS patients with an AUROC of 0.725 [0.633-0.810].

1000G Variants

Supplementary Table 2 XLSX file 490K, Illumina GoldenGate Methylation Standard Cancer Panel I Target CpG Annotations and differential methylation significance values for group comparisons listed in Table

Demographic and clinical characteristics of patients treated with belinostat and panobinostat with archived tumor tissue available for study.

LFS Variants

Exposures to avoid prior to metanephrine testing

Von Hippel–Lindau disease (vHL) is a hereditary tumor predisposition syndrome that places affected individuals at risk for multiple tumors, which are predominantly benign and generally occur in the central nervous system or abdomen. Although the majority of tumors occur in adults, children and adolescents with the condition develop a significant proportion of vHL manifestations and are vulnerable to delayed tumor detection and their sequelae. Although multiple tumor screening paradigms are currently being utilized for patients with vHL, surveillance should be reassessed as the available relevant clinical information continues to expand. We propose a new vHL screening paradigm similar to existing approaches, with important modifications for some tumor types, placing an emphasis on risks in childhood. This includes advancement in the timing of surveillance initiation and increased frequency of screening evaluations. Another neuroendocrine-related familial condition is the rapidly expanding hereditary paraganglioma and pheochromocytoma syndrome (HPP). The tumor spectrum for patients with HPP syndrome includes paragangliomas, pheochromocytomas, renal cancer, and gastrointestinal stromal tumors. The majority of patients with HPP syndrome harbor an underlying variant in one of the SHDx genes (SDHA, SDHB, SDHC, SDHD, SDHA, and SDHAF2), although other genes also have been described (MAX and TMEM127). Annual screening for elevated plasma or urine markers along with complete blood count and biennial whole-body MRI accompanied by focal neck MRI is recommended for older children and adults with HPP syndrome to detect tumors early and to decrease morbidity and mortality from HPP-related tumors. Clin Cancer Res; 23(12); e68–e75. ©2017 AACR.See all articles in the online-only CCR Pediatric Oncology Series.

Supplementary Table 1 from Oncogenic BRAF Mutation with CDKN2A Inactivation Is Characteristic of a Subset of Pediatric Malignant Astrocytomas

Supplemental Table 3. Primer sequences.