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Appendix

Figure S1. Sample flow-chart describing the number of biological samples and clinical data obtained through an international multi-institutional effort and the distribution of samples in each microarray

Table S1-S3. S1Choroid plexus tumor chromosomal instability reported in literature S2-Characterization of patient and sample cohort used in analysis S3 Frequency of TP53 mutations in CPTs and characterization of mutation types. Abbreviations: CPC: choroid plexus carcinoma, CPP: choroid plexus papilloma, aCPP: atypical choroid plexus papilloma, SH3: SRC Homology 3 Domain

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Supplementary Figure 4 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

Supplementary Figure Legends 1-7 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

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Supplementary Figure 3 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

Supplementary Figure 1 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

Supplementary Figure 2 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

Supplementary Tables 1-7 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

Osteosarcomas are copy number alteration (CNA)–rich malignant bone tumors. Using microarrays, fluorescence in situ hybridization, and quantitative PCR, we characterize a focal region of chr3q13.31 (osteo3q13.31) harboring CNAs in 80% of osteosarcomas. As such, osteo3q13.31 is the most altered region in osteosarcoma and contests the view that CNAs in osteosarcoma are nonrecurrent. Most (67%) osteo3q13.31 CNAs are deletions, with 75% of these monoallelic and frequently accompanied by loss of heterozygosity (LOH) in flanking DNA. Notably, these CNAs often involve the noncoding RNAs LOC285194 and BC040587 and, in some cases, a tumor suppressor gene that encodes the limbic system-associated membrane protein (LSAMP). Ubiquitous changes occur in these genes in osteosarcoma, usually involving loss of expression. Underscoring their functional significance, expression of these genes is correlated with the presence of osteo3q13.31 CNAs. Focal osteo3q13.31 CNAs and LOH are also common in cell lines from other cancers, identifying osteo3q13.31 as a generalized candidate region for tumor suppressor genes. Osteo3q13.31 genes may function as a unit, given significant correlation in their expression despite the great genetic distances between them. In support of this notion, depleting either LSAMP or LOC285194 promoted proliferation of normal osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1. Moreover, genetic deletions of LOC285194 or BC040587 were also associated with poor survival of osteosarcoma patients. Our findings identify osteo3q13.31 as a novel region of cooperatively acting tumor suppressor genes. Cancer Res; 70(1); 160–71

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Supplementary Figures 5-7 from Recurrent Focal Copy-Number Changes and Loss of Heterozygosity Implicate Two Noncoding RNAs and One Tumor Suppressor Gene at Chromosome 3q13.31 in Osteosarcoma

Background: Li-Fraumeni syndrome (LFS) is an autosomal dominant cancer predisposition syndrome. Approximately 80% of individuals with LFS harbor a germline TP53 pathogenic variant rendering them susceptible to a wide spectrum of early-onset malignancies. A comprehensive surveillance regimen termed the ‘Toronto Protocol’, has recently been adopted for early tumor detection, demonstrating significant improvement in survival among TP53 pathogenic variant carriers. However, the protocol’s “one-size-fits-all” approach fails to consider an individual patient's risk of cancer. We built a machine learning model that predicts early-onset of primary tumors in LFS by estimating the probability of cancer onset before the age of six years, leveraging patient peripheral blood leukocyte methylation profiles. Methods: We built a gradient-boosted tree model to predict the probability of cancer onset before the age of six using methylation data from 288 TP53 pathogenic variant carriers. An external test set of 82 TP53 pathogenic variant carriers was used to validate our model. To increase the signal-to-noise ratio, methylation probes associated with TP53 status were retained and probes associated with aging were removed. In our study, we were primarily interested in minimizing the false negative rate (i.e. to reduce the number of patients who developed cancer before the age of six but were undetected by our algorithm. Findings: We correctly predicted whether the first tumor will occur before the age of six with an accuracy of 79% in our external test set. Importantly, our model classified 90% of the patients that developed cancer prior to the age of six correctly. In addition, 81% of the individuals without cancer in the external test set were predicted correctly. Interpretation: Our tool provides additional value to clinicians in stratifying patients into low- or high-risk groups of developing early-onset malignancies, and helps inform rational use of clinical surveillance tools for early cancer detection, with the ultimate aim to improve overall patient outcomes. Citation Format: Vallijah Subasri, Benjamin Brew, Lauren Erdman, Tanya Guha, Jordan R. Hansford, Elizabeth Cairney, Carol Portwine, Christine Elser, Jonathan L. Finlay, Kim E. Nichols, Wendy Kohlmann, Noa Alon, Ana Novokmet, Ledia Brunga, Anita Villani, Kelvin C. de Andrade, Payal P. Khincha, Sharon A. Savage, Joshua D. Schiffman, David Malkin, Anna Goldenberg. DNA methylation predicts early onset of primary tumor in patients with Li-Fraumeni syndrome [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 1428.

Germline polymorphic variants in cancer predisposition genes such as TP53 have been shown to impact the risk of premenopausal cancer. Accordingly, the aim of this study was to assess the spectrum of polymorphisms in TP53 and its negative regulatory gene, MDM2 (SNP309:T>G) in patients with premenopausal breast cancer. Our findings in a cohort of 40 female patients demonstrate no significant correlation between the studied polymorphisms and risk of premenopausal breast cancer. Although one polymorphism is found in high frequency in this cohort (rs1800372:A>G, 9.0%), it was not associated with the risk of developing cancer before the age of 35 years in an extended cohort of 1,420 breast cancer cases. Functional studies of the rs1800372:A>G polymorphic allele reveal that it does not affect p53 transactivation function. Further study of variants or mutations in other cancer susceptibility genes is warranted to refine our understanding of the germline contribution to premenopausal breast cancer susceptibility.

Histological grading of choroid plexus tumors (CPTs) remains the best prognostic tool to distinguish between aggressive choroid plexus carcinoma (CPC) and the more benign choroid plexus papilloma (CPP) or atypical choroid plexus papilloma (aCPP); however, these distinctions can be challenging. Standard treatment of CPC is very aggressive and often leads to severe damage to the young child's brain. Therefore, it is crucial to distinguish between CPC and less aggressive entities (CPP or aCPP) to avoid unnecessary exposure of the young patient to neurotoxic therapy. To better stratify CPTs, we utilized DNA methylation (DNAm) to identify prognostic epigenetic biomarkers for CPCs.We obtained DNA methylation profiles of 34 CPTs using the HumanMethylation450 BeadChip from Illumina, and the data was analyzed using the Illumina Genome Studio analysis software. Validation of differentially methylated CpG sites chosen as biomarkers was performed using pyrosequencing analysis on additional 22 CPTs. Sensitivity testing of the CPC DNAm signature was performed on a replication cohort of 61 CPT tumors obtained from Neuropathology, University Hospital Münster, Germany.Generated genome-wide DNAm profiles of CPTs showed significant differences in DNAm between CPCs and the CPPs or aCPPs. The prediction of clinical outcome could be improved by combining the DNAm profile with the mutational status of TP53. CPCs with homozygous TP53 mutations clustered as a group separate from those carrying a heterozygous TP53 mutation or CPCs with wild type TP53 (TP53-wt) and showed the worst survival outcome. Specific DNAm signatures for CPCs revealed AK1, PER2, and PLSCR4 as potential biomarkers for CPC that can be used to improve molecular stratification for diagnosis and treatment.We demonstrate that combining specific DNAm signature for CPCs with histological approaches better differentiate aggressive tumors from those that are not life threatening. These findings have important implications for future prognostic risk prediction in clinical disease management.

Background Histological grading of choroid plexus tumors (CPTs) remains the best prognostic tool to distinguish between aggressive choroid plexus carcinoma (CPC) and the more benign choroid plexus papilloma (CPP) or atypical choroid plexus papilloma (aCPP); however, these distinctions can be challenging. Standard treatment of CPC is very aggressive and often leads to severe damage to the young child’s brain. Therefore, it is crucial to distinguish between CPC and less aggressive entities (CPP or aCPP) to avoid unnecessary exposure of the young patient to neurotoxic therapy. To better stratify CPTs, we utilized DNA methylation (DNAm) to identify prognostic epigenetic biomarkers for CPCs. Methods We obtained DNA methylation profiles of 34 CPTs using the HumanMethylation450 BeadChip from Illumina, and the data was analyzed using the Illumina Genome Studio analysis software. Validation of differentially methylated CpG sites chosen as biomarkers was performed using pyrosequencing analysis on additional 22 CPTs. Sensitivity testing of the CPC DNAm signature was performed on a replication cohort of 61 CPT tumors obtained from Neuropathology, University Hospital Münster, Germany. Results Generated genome-wide DNAm profiles of CPTs showed significant differences in DNAm between CPCs and the CPPs or aCPPs. The prediction of clinical outcome could be improved by combining the DNAm profile with the mutational status of TP53. CPCs with homozygous TP53 mutations clustered as a group separate from those carrying a heterozygous TP53 mutation or CPCs with wild type TP53 (TP53-wt) and showed the worst survival outcome. Specific DNAm signatures for CPCs revealed AK1, PER2, and PLSCR4 as potential biomarkers for CPC that can be used to improve molecular stratification for diagnosis and treatment. Conclusions We demonstrate that combining specific DNAm signature for CPCs with histological approaches better differentiate aggressive tumors from those that are not life threatening. These findings have important implications for future prognostic risk prediction in clinical disease management. Electronic supplementary material The online version of this article (10.1186/s13148-019-0708-z) contains supplementary material, which is available to authorized users.

Purpose Although the link between mutant TP53 and human cancer is unequivocal, a significant knowledge gap exists in clinically actionable molecular targets in Li-Fraumeni syndrome (LFS), a highly penetrant cancer predisposition syndrome associated with germline mutations in TP53. This study surveyed the epigenome to identify functionally and clinically relevant novel genes implicated in LFS. Patients and Methods We performed genome-wide methylation analyses of peripheral blood leukocyte DNA in germline TP53 mutation carriers (n = 72) and individuals with TP53 wild type in whom histologically comparable malignancies developed (n = 111). Targeted bisulfite pyrosequencing was performed on a validation cohort of 30 TP53 mutation carriers and 46 patients with TP53 wild type, and candidate sites were evaluated in primary tumors from patients with LFS across multiple histologic tumor types. Results In 183 patients, distinct DNA methylation signatures were associated with deleterious TP53 mutations in peripheral blood leukocytes. TP53-associated DNA methylation marks occurred in genomic regions that harbored p53 binding sites and in genes encoding p53 pathway proteins. Moreover, loss-of-function TP53 mutations were significantly associated with differential methylation at the locus encoding microRNA miR-34A, a key component of the p53 regulatory network (adjusted P < .001), and validated in an independent patient cohort (n = 76, P < .001). Targeted bisulfite pyrosequencing demonstrated that miR-34A was inactivated by hypermethylation across many histologic types of primary tumors from patients with LFS. Moreover, miR-34A tumor hypermethylation was associated with decreased overall survival in a cohort of 29 patients with choroid plexus carcinomas, a characteristic LFS tumor (P < .05). Conclusion Epigenetic dysregulation of miR-34A may comprise an important path in TP53-associated cancer predisposition and represents a therapeutically actionable target with potential clinical relevance.

INTRODUCTION Gene fusions are often disease-defining events in cancer. The mutational processes that give rise to fusions, their timing relative to initial diagnosis, and whether they change at relapse are largely unknown. Mutational processes leave distinct marks in the tumor genome, meaning that DNA sequencing can be used to reconstruct how fusions are generated. A prototypical fusion-driven tumor is Ewing sarcoma (ES), a bone cancer predominantly affecting children and young adults. ES is defined by fusions involving EWSR1 , a gene encoding an RNA binding protein, and genes encoding E26 transformation-specific (ETS) transcription factors such as FLI1 . We sought to reconstruct the genomic events that give rise to EWSR1-ETS fusions in ES and chart their evolution from diagnosis to relapse. RATIONALE We studied the processes underpinning gene fusions in ES using the whole-genome sequences of 124 primary tumors. We determined the timing of the emergence of EWSR1 fusions relative to other mutations. To measure ongoing mutation rates and evolutionary trajectories of ES, we studied the genomes of primary tumors, tumors at relapse, and metastatic tumors. RESULTS We found that EWSR1-ETS , the key ES fusion, arises in 42% of cases via complex, loop-like rearrangements called chromoplexy, rather than by simple reciprocal translocations. Similar loops forming canonical fusions were found in three other sarcoma types. Timing the emergence of loops revealed that they occur as bursts in early replicating DNA, as a primary event in ES development. Additional gene disruptions are generated concurrently with the fusions within the loops. Chromoplexy-generated EWSR1 fusions appear to be associated with an aggressive form of the disease and a higher chance of relapse. Numerous mutations present in every cell of the primary were absent at relapse, demonstrating that the primary and relapsed diseases evolved independently. This divergence occurs after formation of an ancestral clone harboring EWSR1 fusions. Importantly, we determined that divergence of the primary tumor and the future relapsed tumor occurs 1 to 2 years before initial diagnosis, as estimated from the number of cell division–associated mutations. CONCLUSION Our findings provide insights into the pathogenesis and natural history of human sarcomas. They reveal complex DNA rearrangements to be a mutational process underpinning gene fusions in a large proportion of ES. Similar observations in other fusion-defined sarcoma types indicate that this process operates more generally. Such complex rearrangements occur preferentially in early replicating and transcriptionally active genomic regions, as evidenced by the additional genes disrupted. EWSR1 fusions arising from chromoplexy correlated with worse clinical outcomes. Formation of the EWSR1 fusion genes is a primary event in the life history of ES. We found evidence of a latency period between this seeding event and diagnosis. This is in keeping with the often-indolent nature of symptoms before clinical disease presentation.

Purpose: To investigate molecular alterations in choroid plexus tumors (CPT) using a genome-wide high-throughput approach to identify diagnostic and prognostic signatures that will refine tumor stratification and guide therapeutic options. Experimental Design: One hundred CPTs were obtained from a multi-institutional tissue and clinical database. Copy-number (CN), DNA methylation, and gene expression signatures were assessed for 74, 36, and 40 samples, respectively. Molecular subgroups were correlated with clinical parameters and outcomes. Results: Unique molecular signatures distinguished choroid plexus carcinomas (CPC) from choroid plexus papillomas (CPP) and atypical choroid plexus papillomas (aCPP); however, no significantly distinct molecular alterations between CPPs and aCPPs were observed. Allele-specific CN analysis of CPCs revealed two novel subgroups according to DNA content: hypodiploid and hyperdiploid CPCs. Hyperdiploid CPCs exhibited recurrent acquired uniparental disomy events. Somatic mutations in TP53 were observed in 60% of CPCs. Investigating the number of mutated copies of p53 per sample revealed a high-risk group of patients with CPC carrying two copies of mutant p53, who exhibited poor 5-year event-free (EFS) and overall survival (OS) compared with patients with CPC carrying one copy of mutant p53 (OS: 14.3%, 95% confidence interval, 0.71%–46.5% vs. 66.7%, 28.2%–87.8%, respectively, P = 0.04; EFS: 0% vs. 44.4%, 13.6%–71.9%, respectively, P = 0.03). CPPs and aCPPs exhibited favorable survival. Discussion: Our data demonstrate that differences in CN, gene expression, and DNA methylation signatures distinguish CPCs from CPPs and aCPPs; however, molecular similarities among the papillomas suggest that these two histologic subgroups are indeed a single molecular entity. A greater number of copies of mutated TP53 were significantly associated to increased tumor aggressiveness and a worse survival outcome in CPCs. Collectively, these findings will facilitate stratified approaches to the clinical management of CPTs. Clin Cancer Res; 21(1); 184–92. ©2014 AACR.

BACKGROUND Li-Fraumeni syndrome is a rare genetic cancer predisposition syndrome caused by germline TP53 mutations. Up to 20% of mutation carriers develop cancer during childhood. The benefits of TP53 mutation testing of children are a matter of debate and knowledge of parent decision-making around such testing is limited. The current study examined how parents make decisions regarding TP53 testing for their children. METHODS Families offered and those pursuing TP53 testing for their children were identified across the study sites. Qualitative interviews with 46 parents (39 families) were analyzed to describe decision-making styles and perceived advantages and disadvantages of testing. RESULTS TP53 mutation testing uptake was high (92%). Three decision-making styles emerged. Automatic decisions (44% of decisions) involved little thought and identified immediate benefit(s) in testing (100% pursued testing). Considered decisions (49%) weighed the risks and benefits but were made easily (77% pursued testing). Deliberated decisions (6%) were difficult and focused on psychosocial concerns (25% pursued testing). Perceived advantages of testing included promoting child health, satisfying a “need to know,” understanding why cancer(s) occurred, suggesting family member risk, and benefiting research. Disadvantages included psychosocial risks and privacy/discrimination/insurance issues. CONCLUSIONS Although empirical evidence regarding the benefits and risks of TP53 testing during childhood are lacking, the majority of parents in the current study decided easily in favor of testing and perceived a range of advantages. The authors conclude that in the context of a clinical diagnosis of Li-Fraumeni syndrome, parents should continue to be offered TP53 testing for their children, counseled regarding potential risks and benefits, and supported in their decision-making process. Cancer 2015;121:286–93. © 2014 American Cancer Society.

Li-Fraumeni syndrome (LFS) is a familial cancer predisposition syndrome (CPS) caused by germline mutations in TP53, associated with a high frequency of sarcomas, breast cancers, adrenocortical carcinomas and CNS tumours. Recently, our group and others have identified distinct mutational signatures, defined by the spectrum and context of somatic mutations, in tumours arising in individuals with another CPS, constitutional mismatch repair deficiency (CMMRD). CMMRD tumours frequently harbor somatic mutations disrupting the proofreading function of the DNA polymerases POLE and POLD1, which consequently leads to an ultra-hypermutant cancer genome, with early MMR mutational signatures and late POLE/POLD1 mutational signatures. This ultrahypermutant tumor phenotype is essentially diagnostic for the syndrome and provides a rational for immune checkpoint inhibitors which have shown success in this context. Based on our results in CMMRD tumors we hypothesize that LFS tumours might likewise harbor distinct mutational events and/or evolutionary dynamics from sporadic tumours of the same histiotype. Although several cancer genomics landscape studies have included a handful of tumours from LFS patients, to our knowledge no study to date has attempted to comprehensively characterize the cancer genomes of LFS patients. To investigate the somatic mutational events driving tumourigenesis in LFS we performed whole-genome sequencing (WGS) analysis of 22 tumours derived from patients with pathogenic germline TP53 mutations. Tumours from germline TP53 wildtype patients were analyzed by the same methods to serve as a control data set. For each tumour sample, where possible, we performed WGS on multiple spatially distinct micro-dissected tumour regions in order to reconstruct the evolutionary history of each cancer. Somatic variant calling was performed at high sensitivity using in silico reconstructed high-depth bulk WGS (80-120X coverage), with somatic mutations, structural variants, copy number alterations, mutational signatures and subclones identified using MuTect2, delly, battenberg, SigProfiler and phyloWGS respectively. High confidence variants were identified using in-house designed filtering pipelines. The resulting analyses reveals the life history of LFS cancers is marked by a high frequency of early catastrophic genomic rearrangement events, a diverse range of somatic driver events and in at least some cases marked intratumoural spatial heterogeneity of CNVs and SNVs. Citation Format: Nicholas Light, Matthew Zatzman, Nathaniel Anderson, Vallijah Subasri, Mehdi Layeghifard, Ana Novokmet, James Tran, Richard de Borja, Fabio Fuligni, Joshua Schiffman, David Malkin, Adam Shlien. The genomic landscape and clonal evolution of tumours arising in TP53 mutation carriers [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 3666.

BACKGROUND Rhabdomyosarcoma (RMS) represents a diverse category of myogenic malignancies with marked differences in molecular alterations and histology. This study examines the question if RMS predisposition due to germline TP53 mutations correlates with certain RMS histologies. METHODS The histology of RMS tumors diagnosed in 8 consecutive children with TP53 germline mutations was reviewed retrospectively. In addition, germline TP53 mutation analysis was performed in 7 children with anaplastic RMS (anRMS) and previously unknown TP53 status. RESULTS RMS tumors diagnosed in 11 TP53 germline mutation carriers all exhibited nonalveolar, anaplastic histology as evidenced by the presence of enlarged hyperchromatic nuclei with or without atypical mitotic figures. Anaplastic RMS was the first malignant diagnosis for all TP53 germline mutation carriers in this cohort, and median age at diagnosis was 40 months (mean, 40 months ± 15 months; range, 19-67 months). The overall frequency of TP53 germline mutations was 73% (11 of 15 children) in pediatric patients with anRMS. The frequency of TP53 germline mutations in children with anRMS was 100% (5 of 5 children) for those with a family cancer history consistent with Li-Fraumeni syndrome (LFS), and 80% (4 of 5 children) for those without an LFS cancer phenotype. CONCLUSIONS Individuals harboring germline TP53 mutations are predisposed to develop anRMS at a young age. If future studies in larger anRMS cohorts confirm the findings of this study, the current Chompret criteria for LFS should be extended to include children with anRMS irrespective of family history. Cancer 2014;120:1068–1075. © 2013 American Cancer Society.

Summary Background Carriers of a germline TP53 pathogenic variant have a substantial lifetime risk of developing cancer. In 2011, we did a prospective observational study of members of families who chose to either undergo a comprehensive surveillance protocol for individuals with Li-Fraumeni syndrome or not. We sought to update our assessment of and modify the surveillance protocol, so in this study we report both longer follow-up of these patients and additional patients who underwent surveillance, as well as update the originally presented surveillance protocol. Methods A clinical surveillance protocol using physical examination and frequent biochemical and imaging studies (consisting of whole-body MRI, brain MRI, breast MRI, mammography, abdominal and pelvic ultrasound, and colonoscopy) was introduced at three tertiary care centres in Canada and the USA on Jan 1, 2004, for carriers of TP53 pathogenic variants. After confirmation of TP53 mutation, participants either chose to undergo surveillance or chose not to undergo surveillance. Patients could cross over between groups at any time. The primary outcome measure was detection of asymptomatic tumours by surveillance investigations. The secondary outcome measure was 5 year overall survival established from a tumour diagnosed symptomatically (in the non-surveillance group) versus one diagnosed by surveillance. We completed survival analyses using an as-treated approach. Findings Between Jan 1, 2004, and July 1, 2015, we identified 89 carriers of TP53 pathogenic variants in 39 unrelated families, of whom 40 (45%) agreed to surveillance and 49 (55%) declined surveillance. 19 (21%) patients crossed over from the non-surveillance to the surveillance group, giving a total of 59 (66%) individuals undergoing surveillance for a median of 32 months (IQR 12–87). 40 asymptomatic tumours have been detected in 19 (32%) of 59 patients who underwent surveillance. Two additional cancers were diagnosed between surveillance assessments (false negatives) and two biopsied lesions were non-neoplastic entities on pathological review (false positives). Among the 49 individuals who initially declined surveillance, 61 symptomatic tumours were diagnosed in 43 (88%) patients. 21 (49%) of the 43 individuals not on surveillance who developed cancer were alive compared with 16 (84%) of the 19 individuals undergoing surveillance who developed cancer (p=0·012) after a median follow-up of 46 months (IQR 22–72) for those not on surveillance and 38 months (12–86) for those on surveillance. 5 year overall survival was 88·8% (95% CI 78·7–100) in the surveillance group and 59·6% (47·2–75·2) in the non-surveillance group (p=0·0132). Interpretation Our findings show that long-term compliance with a comprehensive surveillance protocol for early tumour detection in individuals with pathogenic TP53 variants is feasible and that early tumour detection through surveillance is associated with improved long-term survival. Incorporation of this approach into clinical management of these patients should be considered. Funding Canadian Institutes for Heath Research, Canadian Cancer Society, Terry Fox Research Institute, SickKids Foundation, and Soccer for Hope Foundation.

Li-Fraumeni Syndrome (LFS) is a rare autosomal dominant familial cancer syndrome, characterized by multiple malignancies and frequent germline alterations in TP53. In this study, we highlight four unclassified exonic TP53 variants detected in patients with a suspected diagnosis of LFS. Most intriguing was the discovery of a "super-transactivation" variant within Exon 10 of TP53 (c.1079G>T/p.G360V). Functional analysis of this novel variant revealed a paradoxical "super-transactivation" effect on tp53 response elements and a corresponding tumor suppressive effect on colony formation and apoptosis. While unlikely to be disease-causing, we propose that this variant may represent a novel tp53 polymorphism and potential phenotypic modifier in LFS. In the future, the enhanced transactivation effects of p.G360V-tp53 may also prove useful in designing more efficacious tp53-based gene therapies.

Summary Background Individuals with Li-Fraumeni syndrome have a high lifetime risk of developing cancer. We assessed the feasibility and potential clinical effect of a comprehensive surveillance protocol in asymptomatic TP53 mutation carriers in families with this syndrome. Methods We implemented a clinical surveillance protocol, using frequent biochemical and imaging studies, for asymptomatic TP53 mutation carriers on Jan 1, 2004, and did a prospective observational study of members of eight families with Li-Fraumeni syndrome who either chose to undergo surveillance or chose not to undergo surveillance. The primary outcome measure was detection of new cancers. The secondary outcome measure was overall survival. Findings As of Nov 1, 2010, 33 TP53 mutation carriers were identified, 18 of whom underwent surveillance. The surveillance protocol detected ten asymptomatic tumours in seven patients, including small, high-grade tumours and low-grade or premalignant tumours. All seven mutation carriers were alive after a median follow-up of 24 months (IQR 22–65 months). 12 high-grade, high-stage tumours developed in 10 individuals in the non-surveillance group, two of whom (20%) were alive at the end of follow-up (p=0·0417 for comparison with survival in the surveillance group). 3-year overall survival was 100% in the surveillance group and 21% (95% CI 4–48%) in the non-surveillance group (p=0·0155). Interpretation Our findings show the feasibility of a clinical surveillance protocol for the detection of asymptomatic neoplasms in individuals with germline TP53 mutations. This strategy offers a management option for affected individuals, and its benefits lend support to the use of early genetic testing of at-risk individuals and families. Funding Canadian Cancer Society Research Institute, Canadian Institutes of Health Research, SickKids Foundation, and Soccer for Hope.

Osteosarcomas are copy number alteration (CNA)–rich malignant bone tumors. Using microarrays, fluorescence in situ hybridization, and quantitative PCR, we characterize a focal region of chr3q13.31 (osteo3q13.31) harboring CNAs in 80% of osteosarcomas. As such, osteo3q13.31 is the most altered region in osteosarcoma and contests the view that CNAs in osteosarcoma are nonrecurrent. Most (67%) osteo3q13.31 CNAs are deletions, with 75% of these monoallelic and frequently accompanied by loss of heterozygosity (LOH) in flanking DNA. Notably, these CNAs often involve the noncoding RNAs LOC285194 and BC040587 and, in some cases, a tumor suppressor gene that encodes the limbic system-associated membrane protein (LSAMP). Ubiquitous changes occur in these genes in osteosarcoma, usually involving loss of expression. Underscoring their functional significance, expression of these genes is correlated with the presence of osteo3q13.31 CNAs. Focal osteo3q13.31 CNAs and LOH are also common in cell lines from other cancers, identifying osteo3q13.31 as a generalized candidate region for tumor suppressor genes. Osteo3q13.31 genes may function as a unit, given significant correlation in their expression despite the great genetic distances between them. In support of this notion, depleting either LSAMP or LOC285194 promoted proliferation of normal osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1. Moreover, genetic deletions of LOC285194 or BC040587 were also associated with poor survival of osteosarcoma patients. Our findings identify osteo3q13.31 as a novel region of cooperatively acting tumor suppressor genes. Cancer Res; 70(1); 160–71

Purpose Adrenocortical carcinoma (ACC) is a rare pediatric malignancy. It occurs in excess among individuals with the Li-Fraumeni syndrome, which results primarily from germline mutations in the TP53 gene. Prior series exploring frequencies of germline TP53 mutation among children with ACC have been small, geographically limited, or subject to referral bias. The functional consequence of mutations has not been related to phenotype. We provide a genotype-phenotype analysis of TP53 mutations in pediatric ACC and propose a model for tissue-specific effects based on adrenocortical ontogeny. Patients and Methods Eighty-eight consecutive, unrelated children with ACC, unselected for family history, underwent germline TP53 sequencing. Rate and distribution of mutations were identified. Functional analysis was performed for novel TP53 variants. Correlation with the International Agency for Research on Cancer p53 database further delineated mutational distribution, association with family history, and risk for multiple primary malignancies (MPMs). Results Germline mutations were present in 50% of children. These mutations did not correspond to the conventional hotspot mutations. There was a wide range of mutant protein function. Patients bearing alleles encoding protein with higher functionality were less likely to have a strong family cancer history, whereas those with greater loss of function had MPMs and/or positive family history. In patients with MPMs, ACC was the most frequent initial malignancy. Finally, we demonstrated age-dependent rates of TP53 mutation positivity. Conclusion TP53 mutations are prevalent in children with ACC but decline with age. Mutations result in a broad spectrum of functional loss. Effect of individual mutations may predict carrier and familial disease penetrance with potentially broad implications for clinical surveillance and counseling.

Introduction: Li Fraumeni Syndrome (LFS) is a rare hereditary genetic cancer predisposition syndrome. Germline mutations of the TP53 tumor suppressor gene are the underlying cause in >80% of patients with LFS, and are associated with an increased risk of second tumors and a spectrum of early onset cancers, even in the absence of a family history of cancer. We have previously developed and implemented a comprehensive life-long clinical surveillance protocol for individuals with a germline TP53 mutation. We set out to make this screening process more targeted by building a predictive model of age of onset. We accomplished this goal by implementing machine learning methods on germline methylation data. Methods: We made use of the Toronto Hospital for Sick Children (SickKids) LFS family cohort in our predictive model of age of onset. In all, we have 74 patients with germline methylation data, consisting of ~450,000 probe sites. We subset this data by identifying probes that fall into differentially methylated regions between LFS and cancer patients with wild-type TP53. The probes identified in these regions were used in our predictive model of age of onset. Because age of sample collection was highly correlated with age of onset (r2 ~ .90), we corrected for confounding using a strategy that is two-fold: (1) we extracted the variation of each probe that is independent of the age of sample collection (the residual after regressing on the age of sample collection) and use these as predictors in our model, and (2) we test our models on the task of predicting the age of sample collection for LFS patients that do not have cancer. The former provided us with more robust predictions while the latter verified that we are in fact predicting age of onset, rather than simply predicting age at which the sample was collected. Results: Our machine learning model was able to achieve 86% correlation between true and predicted values of the age of onset. Additionally, we have tested the ability of our models to predict whether an individual will be diagnosed before or after the age of 4. Our classification machine learning model achieved 91% accuracy on average. We verified that our model does not simply predict age of sample collection by using our cohort of LFS patients that do not have cancer (n = 37). The distribution of the age of sample collection matched those of the patients used in our model. The model has no predictive power on the age of sample collection, thus confirming that our model is highly predictive of the age of cancer onset in LFS TP53 Mutation patients. Conclusions: We identified two predictive models for age of cancer onset in LFS patients that achieve high accuracy, both when predicting the age of onset as a continuous variable (86% correlation) and whether cancer onset will occur before or after the age of 4 (91% accuracy). Our model will assist clinicians in targeting high risk patients for screening, lower the cost of treatment, and raise the likelihood of survival among LFS patients. Citation Format: Benjamin M. Brew, David Malkin, Lauren Erdman, Andrea Doria, Jason Berman, Adam Shlien, Tanya Guha, Ana Novokmet, Anna Goldenberg. Methylation accurately predicts age of cancer onset in patients with Li Fraumeni Syndrome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 973. doi:10.1158/1538-7445.AM2017-973

Li-Fraumeni syndrome is a rare genetic cancer predisposition syndrome caused by germline TP53 mutations. Up to 20% of mutation carriers develop cancer during childhood. The benefits of TP53 mutation testing of children are a matter of debate and knowledge of parent decision-making around such testing is limited. The current study examined how parents make decisions regarding TP53 testing for their children.Families offered and those pursuing TP53 testing for their children were identified across the study sites. Qualitative interviews with 46 parents (39 families) were analyzed to describe decision-making styles and perceived advantages and disadvantages of testing.TP53 mutation testing uptake was high (92%). Three decision-making styles emerged. Automatic decisions (44% of decisions) involved little thought and identified immediate benefit(s) in testing (100% pursued testing). Considered decisions (49%) weighed the risks and benefits but were made easily (77% pursued testing). Deliberated decisions (6%) were difficult and focused on psychosocial concerns (25% pursued testing). Perceived advantages of testing included promoting child health, satisfying a "need to know," understanding why cancer(s) occurred, suggesting family member risk, and benefiting research. Disadvantages included psychosocial risks and privacy/discrimination/insurance issues.Although empirical evidence regarding the benefits and risks of TP53 testing during childhood are lacking, the majority of parents in the current study decided easily in favor of testing and perceived a range of advantages. The authors conclude that in the context of a clinical diagnosis of Li-Fraumeni syndrome, parents should continue to be offered TP53 testing for their children, counseled regarding potential risks and benefits, and supported in their decision-making process.

Choroid plexus tumors (CPTs) are rare neoplasms of the central nervous system most commonly found in the pediatric population. CPTs represent 1- 4% of all childhood brain tumors, with 10- 20% occurring during the first year of life. Within this family of tumors, choroid plexus carcinoma (CPC) is the malignant neoplasm which is categorized as a grade III tumor by the WHO. Choroid plexus papilloma (CPP) is a benign form classified as a grade I tumor, and atypical choroid plexus papilloma (aCPP) as a grade II tumor. Distinction between these tumor subtypes is essential for treatment stratification. Previous studies performed in our laboratory suggest that CPTs are highly unstable and harbor unique patterns of chromosome-wide gains and losses. To better understand the complexities of tumor biology of CPTs as well as to identify better molecular biomarkers to distinguish between aggressive and benign forms of CPTs we performed a genome-wide DNA methylation study using Illumina Human Methylation450 BeadChip. We analyzed genome-wide DNA methylation profiles from 34 CPT (14 CPCs, 5 aCPPs and 15 CPPs) samples. Differential DNA methylation analysis did not identify significant differences between aCPPs and CPPs, therefore we explored CPC-specific DNA methylation signature in comparison to CPPs. Using a median beta value difference of 0.3 or greater and an FDR adjusted p-value Citation Format: Malgorzata Pienkowska, Sanaa Choufani, Andrei Turinsky, Diana Merino, Ana Novokmet, Michael Brudno, Rosanna Weksberg, Adam Shlien, Cynthia Hawkins, Eric Bouffet, Uri Tabori, Richard Gilbertson, David Malkin. Investigating the role of DNA methylation in pediatric choroid plexus tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2789.

Li-Fraumeni Syndrome (LFS) is a rare autosomal dominant familial cancer syndrome, characterized by multiple malignancies and frequent germline alterations in TP53. In this study, we highlight four unclassified exonic p53 variants detected in patients with a suspected diagnosis of LFS. We report for the first time the discovery of two novel functional variants in codons 191(c.572C>G; p.P191R) and 360 (c.1079G>T; p.G360V), located, respectively, in the DNA binding domain and in a linker region near the tetramerization domain of TP53. Our data revealed that while the P191R variant decreased the transactivation levels of several p53 targets, it failed to segregate with the disease state. The G360V variant, on the other hand, behaved in a paradoxical fashion by causing a stark upregulation in the activity of several p53 response elements. This tumor suppressive effect was also observed at the level of colony formation and c-Caspase 3 activation. While unlikely to be disease-causing, we propose that these variants may represent novel p53 polymorphisms and potential phenotypic modifiers in LFS. In the future, the enhanced transactivation effects of G360V-p53 may also prove useful in designing more efficacious p53-based gene therapies. Citation Format: Badr Id Said, Han Kim, James Tran, Ana Novokmet, David Malkin. Super-transactivation TP53 variant in the germline of a family with Li-Fraumeni variant. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 792.

It is well known that early-onset breast cancer may be due to an inherited predisposition. When evaluating women diagnosed with breast cancer under age 30, two important syndromes are typically considered: Hereditary Breast and Ovarian Cancer Syndrome and Li-Fraumeni syndrome. Many women are offered genetic testing for mutations in the BRCA1 and BRCA2 genes; however, few are offered genetic testing for mutations in the TP53 gene. There is a concern that overly restrictive testing of TP53 may fail to recognize families with Li-Fraumeni syndrome. We reviewed the genetic test results and family histories of all women with early-onset breast cancer who had genetic testing of the TP53 gene at the Toronto Hospital for Sick Children. Of the 28 women tested, six (33.3 %) had a mutation in the TP53 gene; a mutation was found in 7.7 % of women who did not meet current criteria for Li-Fraumeni syndrome. By reviewing similar data published between 2000 and 2011, we estimate that 5–8 % of women diagnosed with early-onset breast cancer, and who have a negative family history, may have a mutation in the TP53 gene. Given the potential benefits versus harms of this testing, we discuss the option of simultaneous testing of all three genes (BRCA1, BRCA2, and TP53) for women diagnosed with breast cancer before age 30.

Although monoallelic expression (MAE) is a frequent genomic event in normal tissues, its role in tumorigenesis remains unclear. Here we carried out single-nucleotide polymorphism arrays on DNA and RNA from a large cohort of pediatric and adult brain tumor tissues to determine the genome-wide rate of MAE, its role in specific cancer-related genes, and the clinical consequences of MAE in brain tumors. We also used targeted genotyping to examine the role of tumor-related genes in brain tumor development and specifically examined the clinical consequences of MAE at TP53 and IDH1. The genome-wide rate of tumor MAE was higher than in previously described normal tissue and increased with specific tumor grade. Oncogenes, but not tumor suppressors, exhibited significantly higher MAE in high-grade compared with low-grade tumors. This method identified nine novel genes highly associated with MAE. Within cancer-related genes, MAE was gene specific; hTERT was most significantly affected, with a higher frequency of MAE in adult and advanced tumors. Clinically, MAE at TP53 exists only in mutated tumors and increases with tumor aggressiveness. MAE toward the normal allele at IDH1 conferred worse survival even in IDH1 mutated tumors. Taken together, our findings suggest that MAE is tumor and gene specific, frequent in brain tumor subtypes, and may be associated with tumor progression/aggressiveness. Further exploration of MAE at relevant genes may contribute to better understanding of tumor development and determine survival in brain tumor patients. Cancer Res; 72(3); 636–44. ©2011 AACR.

DNA copy number variations (CNVs) are a significant and ubiquitous source of inherited human genetic variation. However, the importance of CNVs to cancer susceptibility and tumor progression has not yet been explored. Li–Fraumeni syndrome (LFS) is an autosomal dominantly inherited disorder characterized by a strikingly increased risk of early-onset breast cancer, sarcomas, brain tumors and other neoplasms in individuals harboring germline TP53 mutations. Known genetic determinants of LFS do not fully explain the variable clinical phenotype in affected family members. As part of a wider study of CNVs and cancer, we conducted a genome-wide profile of germline CNVs in LFS families. Here, by examining DNA from a large healthy population and an LFS cohort using high-density oligonucleotide arrays, we show that the number of CNVs per genome is well conserved in the healthy population, but strikingly enriched in these cancer-prone individuals. We found a highly significant increase in CNVs among carriers of germline TP53 mutations with a familial cancer history. Furthermore, we identified a remarkable number of genomic regions in which known cancer-related genes coincide with CNVs, in both LFS families and healthy individuals. Germline CNVs may provide a foundation that enables the more dramatic chromosomal changes characteristic of TP53-related tumors to be established. Our results suggest that screening families predisposed to cancer for CNVs may identify individuals with an abnormally high number of these events.

INTRODUCTION: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome that confers a high lifetime risk of developing malignancies, including pre-menopausal breast cancer. Over 80% of LFS patients harbor germline mutations of the TP53 tumor suppressor gene. OBJECTIVE: We sought to determine if the spectrum of TP53 variants, as well as global methylation methylation patterns, correlates with the presence of TP53 mutations or influences cancer risk in pre-menopausal breast cancer patients. METHODS: Constitutional DNA was obtained from peripheral blood lymphocytes (PBL) of 10 patients with pre-menopausal breast cancer harboring germline TP53 mutations, 10 patients diagnosed with pre-menopausal breast cancer that are non-mutation carriers, 15 TP53 mutation carriers who do not have cancer, and 15 healthy female controls. Genome-wide methylation analysis of PBL-derived DNA was performed using the Illumina 450K array. Sanger sequencing of exons 2-11, and flanking intron regions of the TP53 gene was performed. RESULTS: The most commonly observed single nucleotide polymorphism (SNP) in TP53 across all samples was at c.215C>G (p.Pro72Arg) in exon 4 (rs1042522). We observed a G allele frequency of 66.0% in our study group. Three additional low-frequency variants in TP53 were observed: the exon 4: c.108G>A (p.Pro36Pro) variant was found in 2 patients, exon 6: c.639A>G (p.Arg213Arg) was found in 9 patients and intron 9: c.993+12T>C was found in 4 patients. Methylation patterns were not associated with a specific TP53 mutation or variant; however, distinct methylation patterns were observed in TP53 mutation carriers when compared to either TP53 wild-type healthy individuals or patients with early-onset breast cancer. DISCUSSION: This study represents an epigenome-wide analysis and targeted genetic survey of germline TP53 in early-onset, pre-menoupausal breast cancer. As a result of the mutation frequency observed, germline mutations in TP53 may need to be included in the genetic work-up of patients with early-onset breast cancer. Parallel studies of genome-wide methylation demonstrate these patterns may serve as an independent biomarker of early-onset breast cancer risk. Note: This abstract was not presented at the meeting. Citation Format: Nardin Samuel, Mathieu Lemire, Ana Novokmet, Thomas J. Hudson, David Malkin. Impact of TP53 mutations, single nucleotide variants and global methylation patterns on pre-menopausal breast cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4664. doi:10.1158/1538-7445.AM2015-4664

e12546 Background: Carriers of a germline TP53 mutation have a substantial lifetime risk of developing cancer. We sought to update our evaluation of a comprehensive surveillance protocol for indivi...

Aim: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome that confers a high lifetime risk of developing malignancies, including pre-menopausal breast cancer. Although many families with classic LFS phenotypes harbor germline TP53 mutations, approximately 25% do not. We observe a high frequency of polymorphic TP53 variants in LFS patients not found to harbor deleterious mutations. We sought to determine if the spectrum of TP53 variants in pre-menopausal breast cancer patients influences cancer risk. Methods: Germline DNA was obtained from whole blood of 90 ethnically diverse patients diagnosed with pre-menopausal breast cancer, as well as patients diagnosed with breast cancer in addition to another primary malignancy. The median age at first cancer diagnosis was 35 years. Sanger sequencing of exons 2-11, and flanking intron regions of the TP53 gene was performed. HapMap population frequencies for TP53 polymorphisms were used as reference values. Results: Five of 90 patients in our study were found to harbor known deleterious mutations in TP53, consistent with LFS. The most commonly observed single nucleotide polymorphism (SNP) across all samples was the c.215C>G (p.Pro72Arg) in exon 4 (rs1042522). This polymorphism is a common variant that has G allele frequencies of 76.7%, 51.1% and 33.1% in European, Asian (Han Chinese) and Sub-Saharan African populations, respectively. We observe a G allele frequency of 66.0% in our study group. There was no significant difference in age at cancer diagnosis between any of the three genotypic groups (C/C: 33 years; C/G: 34 years: G/G: 36 years). Three additional low-frequency SNPs in TP53 were observed: the exon 4: c.108G>A (p.Pro36Pro) variant was found in 2 patients, exon 6: c.639A>G (p.Arg213Arg) was found in 9 patients and intron 9: c.993+12T>C was found in 4 patients. There was no observed distinct signature of TP53 polymorphism spectrum in patients who developed multiple primary malignancies. Discussion: This study represents a genetic survey of TP53 in pre-menopausal breast cancer patients of diverse ethnicities. TP53 polymorphisms alone were not sufficient to make clinical predictions about age at breast cancer diagnosis. The TP53 polymorphism, rs1042522, was observed in a large proportion of patients in this study, consistent with its reported frequencies in SNP databases. Parallel studies of CpG methylation of the TP53 promoter and shore regions flanking the locus are being conducted to investigate alternate hypotheses linking TP53 and risk of early-onset breast cancer in the absence of germline TP53 mutations. Citation Format: Nardin Samuel, Ana Novokmet, Thomas J. Hudson, David Malkin. Impact of germline TP53 mutations and polymorphisms in women with premenopausal breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 16. doi:10.1158/1538-7445.CANSUSC14-16

Germline TP53 mutations are observed in ∼80% of patients with Li-Fraumeni syndrome (LFS) and a lesser frequency of patients meeting the revised Chompret criteria. A variety of studies are being conducted exploring the role of genetic modifiers on p53 function and their impact in refining the genotype:phenotype correlation of the TP53:LFS relationship. In this presentation, four classical pediatric-onset LFS tumors (choroid plexus carcinoma, medulloblastoma, adrenocortical carcinoma and rhabdomyosarcoma) will be used as examples to demonstrate the molecular nuances of this complex relationship. A spectrum of findings will be outlined that support the notion that presence of a germline TP53 mutation in the context of different modifying effects of intragenic TP53 polymorphism as well as variants in genes and proteins that play important roles in the p53 network confers certain biological features to the presentation of these tumors. Biological mechanisms will be discussed and strategies for early cancer detection will be presented. Citation Format: Jonathan Wasserman, Anita Villani, Nardin Samuel, Diana Merino, Ana Novokmet, Margaret Pienkowska, Badr IdSaid, David Malkin. Li-Fraumeni syndrome: p53 and beyond. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA10. doi:10.1158/1538-7445.CANSUSC14-IA10

Background: Li-Fraumeni syndrome (LFS) is characterized by marked clinical heterogeneity in site and age of cancer onset. The prolyl-isomerase, Pin1 (peptidylprolyl cis/trans isomerase, NIMA-interacting 1), has been shown to serve a fundamental role in mutant TP53 oncogenic gain of function and the promotion of aggressive cancer phenotypes. We sought to determine whether alterations in PIN1 modify the phenotype of patients with germline mutations in TP53. Methods: The PIN1 promoter and coding regions were sequenced from peripheral blood lymphocyte samples of 107 individuals with Li-Fraumeni syndrome and documented TP53 mutations. The expression and activity levels of PIN1 were determined in paired patient-derived lymphoblastoid cell lines. Results: Multivariate analysis of PIN1 variants with TP53 mutation status and selected clinical parameters is in progress. A comprehensive summary of observed associations will be presented. Conclusions: Demonstration of a modifying effect of PIN1 on the cancer phenotype conferred by a germline TP53 mutation may aid in the management of patients with Li-Fraumeni syndrome, including the implementation of patient-specific modifications of surveillance strategies for pre-symptomatic tumor detection. Citation Format: Anita Villani, Ana Novokmet, David Malkin. Investigating PIN1 as a genetic modifier in Li-Fraumeni syndrome. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 34. doi:10.1158/1538-7445.CANSUSC14-34

Choroid plexus tumors are rare epithelial brain tumors occurring predominantly in early childhood. Understanding molecular events leading to development and progression of these tumors is critical in determining patient treatment and increased survival. Loss of heterozygosity (LOH) is a common alteration observed during carcinogenesis. To investigate somatic genetic abnormalities in different subtypes of choroid plexus cancers and identify LOH hotspots, we utilized SNP 6.0 arrays (Affymetrix). Allele specific copy number analysis of 25 CPCs and 31 CPPs using Chromosome Analysis Suite (Affymetrix) revealed much higher frequency of LOH events throughout the genome in CPCs (8.4/case) compared with CPPs (1.2/case). One LOH hotspot, neutral copy loss of heterozygosity on chromosome 11, was identified in most CPCs but in no CPPs. Chromosome 11 is known to harbor an imprinted cluster that has been associated with growth dysregulation and pediatric cancer. Both maternal (KvDMR1) and paternal (H19DMR) differentially methylated regions overlap this imprinting cluster. Therefore, we investigated whether the observed LOH is related to uniparental disomy (UPD). Bisulfite pyrosequencing on a subset of CPP and CPC were studied for DNA methylation at both H19DMR and KvDMR1. Our data showed a reverse correlation in the degree of DNA methylation between the 2 DMRs in CPCs and CPPs. These data correlate well with the concept of UPD identified through our SNP arrays. Next, we investigated whether the observed higher expression of insulin like growth factor (IGF2) in CPP compared to CPC is associated with DNA methylation aberrations at the imprinted control region (H19 DMR) on 11p15.5. We identified higher DNA methylation at the H19DMR in CPC compared to CPP. Increased activity of the IGF2 gene has been associated with many types of cancer. To investigate the mechanism of CPC distinct IGF2 expression we examined the expression levels of microRNAs (miRNAs). MicroRNAs are often found to be misregulated in human cancer, and they can act posttranscriptionally as either potent oncogenes or tumor suppressor genes. We studied expression of miR-675-3p, miR-483-3p as well as miR-125-a-3p associated with IGF2/H19 imprinted locus. By quantitative RT-PCR, both miR-675 and miR-125 were found to be more significantly upregulated in CPCs than in CPPs whereas miR-483 was expressed at very low levels in CPCs and in higher levels in CPPs compared with normal control tissues . Misregulation of miR-483 was positively correlated with IGF2 levels in both groups. Our data suggest that dysregulation of epigenetic mechanisms contribute to the molecular events leading to tumor development and progression in CPC and may eventually facilitate identification of new diagnostic markers for CPC, as well as new therapeutic targets for this aggressive, lethal tumor. Citation Format: Malgorzata Pienkowska, Sanaa Choufani, Boleslaw Lach, Diana M. Merino, Ana Novokmet, Uri Tabori, Richard Gilbertson, David Malkin. Identification of epigenomic markers on chromosome 11 that distinguish between choroid plexus carcinoma (CPC) and papilloma (CPP). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 661. doi:10.1158/1538-7445.AM2013-661

Introduction: Li-Fraumeni syndrome (LFS) is a rare autosomal dominant disorder associated with germline mutations of the p53 gene. LFS is associated with early age of onset of breast cancer in women, childhood sarcomas and other malignancies. Previous studies have suggested potential correlations between p53 mutation type and clinical cancer phenotypes. For example, missense mutations within the DNA binding loop were found to be associated with higher prevalence of brain tumors and breast cancer whereas missense mutations outside the DNA binding loops were associated with higher prevalence of adrenocortical carcinoma. Through our Cancer Genetics Program (CGP), we have ascertained and conducted p53 mutation analysis on LFS patients/families from 1992 to the present. This represents one of the largest collections in the world. Here we set out to collate and characterize potentially novel genotype:phenotype associations from this database. Methods: TissueMetrix (Artificial Intelligence in Medicine (AIM), Toronto) was used to develop the CGP database. Clincal-pathologic data including family pedigrees, age of tumor onset, tumor type (confirmed with pathology reports where available were collected. From 1992-1998, Sanger sequencing of p53 encompassing the DNA binding domain (exons 5-8) was performed. Since 1998, the entire coding region and additional 50-100 bases spanning intron/exon splice sites have been sequenced and since 2007, MLPA testing has been performed to look for allelic deletion/duplication. Results: p53 mutations have been identified in 110 individuals, of whom 61 (55%) are less than 18years of age. Missense mutations the most common sequence alteration (83/110= 75%). The most commonly observed mutation was the dominant-negative Arg248Gln substitution (n=12). Monoallelic complete or partial gene deletion was observed in 10 cases, several of whom exhibited a non-cancer, developmental delay phenotype. The most common cancer were brain tumors (n=21), of which 10 were choroid plexus carcinomas. ADCC, breast cancer and sarcomas were commonly observed; non-classical LFS tumors, including neuroblastoma, papillary carcinoma of the thyroid and Wilms tumor are also seen. Conclusion: Our program, being situated in a pediatric tertiary centre has an inherent ascertainment bias which likely explains the high frequency of young p53 mutation carriers, as well as a disproportionate prevalence of childhood cancers. Nevertheless, unique correlations emerge from this rich resource including that of p53 deletion and hypotonia/developmental delay, as well as de novo and novel non-coding mutations. These correlations provide the foundation on which to develop practical genetic screening and clinical surveillance approaches to at-risk children and young adults. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1435. doi:1538-7445.AM2012-1435