Diana Merino

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

DNA replication−associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10−13). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.

Methylation of the TERT promoter and risk stratification of childhood brain tumours: an integrative genomic and molecular study

BACKGROUND Identification of robust biomarkers of malignancy and methods to establish disease progression is a major goal in paediatric neuro-oncology. We investigated whether methylation of the TERT promoter can be a biomarker for malignancy and patient outcome in paediatric brain tumours. METHODS For the discovery cohort, we used samples obtained from patients with paediatric brain tumours and individuals with normal brain tissues stored at the German Cancer Research Center (Heidelberg, Germany). We used methylation arrays for genome-wide assessment of DNA. For the validation cohort, we used samples obtained from several tissues for which full clinical and follow-up data were available from two hospitals in Toronto (ON, Canada). We did methylation analysis using quantitative Sequenom and pyrosequencing of an identified region of the TERT promoter. We assessed TERT expression by real-time PCR. To establish whether the biomarker could be used to assess and predict progression, we analysed methylation in paired samples of tumours that transformed from low to high grade and from localised to metastatic, and in choroid plexus tumours of different grades. Finally, we investigated overall survival in patients with posterior fossa ependymomas in which the identified region was hypermethylated or not. All individuals responsible for assays were masked to the outcome of the patients. FINDINGS Analysis of 280 samples in the discovery cohort identified one CpG site (cg11625005) in which 78 (99%) of 79 samples from normal brain tissues and low-grade tumours were not hypermethylated, but 145 (72%) of 201 samples from malignant tumours were hypermethylated (>15% methylated; p<0.0001). Analysis of 68 samples in the validation cohort identified a subset of five CpG sites (henceforth, upstream of the transcription start site [UTSS]) that was hypermethylated in all malignant paediatric brain tumours that expressed TERT but not in normal tissues that did not express TERT (p<0.0001). UTSS had a positive predictive value of 1.00 (95% CI 0.95-1.00) and a negative predictive value of 0.95 (0.87-0.99). In two paired samples of paediatric gliomas, UTSS methylation increased during transformation from low to high grade; it also increased in two paired samples that progressed from localised to metastatic disease. Two of eight atypical papillomas that had high UTSS methylation progressed to carcinomas, while the other six assessed did not progress or require additional treatment. 5-year overall survival was 51% (95% CI 31-71) for 25 patients with hypermethylated UTSS posterior fossa ependymomas and 95% (86-100) for 20 with non-hypermethylated tumours (p=0.0008). 5-year progression-free survival was 86% (68-100) for the 25 patients with non-hypermethylated UTSS tumours and 30% (10-50) for those with hypermethylated tumours (p=0.0008). INTERPRETATION Hypermethylation of the UTSS region in the TERT promoter is associated with TERT expression in cancers. In paediatric brain tumours, UTSS hypermethylation is associated with tumour progression and poor prognosis. This region is easy to amplify, and the assay to establish hypermethylation can be done on most tissues in most clinical laboratories. Therefore the UTSS region is a potentially accessible biomarker for various cancers. FUNDING The Canadian Institute of Health Research and the Terry Fox Foundation.

Molecular Characterization of Choroid Plexus Tumors Reveals Novel Clinically Relevant Subgroups

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.

Spatial heterogeneity in medulloblastoma

Spatial heterogeneity of transcriptional and genetic markers between physically isolated biopsies of a single tumor poses major barriers to the identification of biomarkers and the development of targeted therapies that will be effective against the entire tumor. We analyzed the spatial heterogeneity of multiregional biopsies from 35 patients, using a combination of transcriptomic and genomic profiles. Medulloblastomas (MBs), but not high-grade gliomas (HGGs), demonstrated spatially homogeneous transcriptomes, which allowed for accurate subgrouping of tumors from a single biopsy. Conversely, somatic mutations that affect genes suitable for targeted therapeutics demonstrated high levels of spatial heterogeneity in MB, malignant glioma, and renal cell carcinoma (RCC). Actionable targets found in a single MB biopsy were seldom clonal across the entire tumor, which brings the efficacy of monotherapies against a single target into question. Clinical trials of targeted therapies for MB should first ensure the spatially ubiquitous nature of the target mutation.

Cross-Species Genomics Identifies TAF12, NFYC, and RAD54L as Choroid Plexus Carcinoma Oncogenes

Choroid plexus carcinomas (CPCs) are poorly understood and frequently lethal brain tumors with few treatment options. Using a mouse model of the disease and a large cohort of human CPCs, we performed a cross-species, genome-wide search for oncogenes within syntenic regions of chromosome gain. TAF12, NFYC, and RAD54L co-located on human chromosome 1p32-35.3 and mouse chromosome 4qD1-D3 were identified as oncogenes that are gained in tumors in both species and required for disease initiation and progression. TAF12 and NFYC are transcription factors that regulate the epigenome, whereas RAD54L plays a central role in DNA repair. Our data identify a group of concurrently gained oncogenes that cooperate in the formation of CPC and reveal potential avenues for therapy.

Genome-Wide DNA Methylation Analysis Reveals Epigenetic Dysregulation of MicroRNA-34A in TP53-Associated Cancer Susceptibility

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.

Genome-Wide Association Study to Identify Susceptibility Loci That Modify Radiation-Related Risk for Breast Cancer After Childhood Cancer.

Background Childhood cancer survivors treated with chest-directed radiotherapy have substantially elevated risk for developing breast cancer. Although genetic susceptibility to breast cancer in the general population is well studied, large-scale evaluation of breast cancer susceptibility after chest-directed radiotherapy for childhood cancer is lacking. Methods We conducted a genome-wide association study of breast cancer in female survivors of childhood cancer, pooling two cohorts with detailed treatment data and systematic, long-term follow-up: the Childhood Cancer Survivor Study and St. Jude Lifetime Cohort. The study population comprised 207 survivors who developed breast cancer and 2774 who had not developed any subsequent neoplasm as of last follow-up. Genotyping and subsequent imputation yielded 16 958 466 high-quality variants for analysis. We tested associations in the overall population and in subgroups stratified by receipt of lower than 10 and 10 or higher gray breast radiation exposure. We report P values and pooled per-allele risk estimates from Cox proportional hazards regression models. All statistical tests were two-sided. Results Among survivors who received 10 or higher gray breast radiation exposure, a locus on 1q41 was associated with subsequent breast cancer risk (rs4342822, nearest gene PROX1 , risk allele frequency in control subjects [RAF controls ] = 0.46, hazard ratio = 1.92, 95% confidence interval = 1.49 to 2.44, P = 7.09 × 10 -9 ). Two rare variants also showed potentially promising associations (breast radiation ≥10 gray: rs74949440, 11q23, TAGLN , RAF controls = 0.02, P = 5.84 × 10 -8 ; <10 gray: rs17020562, 1q32.3, RPS6KC1 , RAF controls = 0.0005, P = 6.68 × 10 -8 ). Associations were restricted to these dose subgroups, with consistent findings in the two survivor cohorts. Conclusions Our study provides strong evidence that germline genetics outside high-risk syndromes could modify the effect of radiation exposure on breast cancer risk after childhood cancer.

Basic Science of Pediatric Brain Tumors

Recent advances in genomic and transcriptomic technologies have revolutionized our knowledge of the genetic and molecular basis of pediatric brain tumors. These discoveries have pinpointed novel genes and pathways, identified distinct molecular subgroups, and have led to developments of new mouse models. This chapter details our current understanding of the basic science of pediatric brain tumors, providing an outline of disease mechanisms and potential targets for molecular therapy.

Abstract 2789: Investigating the role of DNA methylation in pediatric choroid plexus tumors

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.

Abstract 3280: Sequencing approaches define the mutation and fusion landscape of choroid plexus carcinomas

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background & Purpose: Choroid plexus carcinomas (CPCs) are aggressive intraventricular tumors accounting for ∼20% of brain tumors in children under 2 years of age. The clinical outcome of CPC patients is variable having an average 5-year overall survival of 50%. A recent study from our group has characterized copy number, DNA methylation and gene expression aberrations in these tumors and identified that a greater number of mutated copies of TP53 is significantly associated with reduced survival. The mutational landscape of these tumors is largely unknown, although this information is crucial to refine the understanding of CPC tumorigenesis and identify novel therapeutic targets. The purpose of this study is to complement the in-depth analysis of the molecular abnormalities driving CPC development by investigating single nucleotide alterations (SNAs), and structural variations (SVs) in CPC DNA and RNA. Methods: A cohort of 29 high quality samples from 25 pediatric CPC patients was used for this study. DNA from 4 blood-tumor pairs and RNA from 27 CPCs were used for whole genome sequencing (WGS) and RNA sequencing (RNA seq), respectively, as part of the Pediatric Cancer Genome Project (PCGP) at St. Jude Childrens Research Hospital, using the Illumina Genome Analyzer IIx or HiSeq platform with 100bp read length. Eighty-two percent of samples were primary tumors, while 17.2% were recurrences. Only one sample had sufficient high quality nucleic acid material to conduct both DNA and RNA sequencing. Results: CPCs exhibited very low levels of SNAs ranging from 1 to 11 SNAs per genome. Copy number analysis using WGS confirmed the high levels of copy number alterations in CPCs. RNAseq analysis revealed that primary and recurrent CPC samples exhibited 100% similarity in fusion breakpoints, suggesting very little clonal divergence in these pediatric brain tumors. A recurrent fusion in chromosome 3 was observed in two independent CPCs (7%). Other fusion events generated in-frame alterations in genes involved in the MAP kinase pathway, such as PPP2R1A, and in apoptosis and cell proliferation pathways. The sample for which WGS and RNAseq was generated exhibited chromothripsis with numerous SVs affecting chromosomes 1 and 19. No other SV events were found in our CPC cohort. Conclusions: CPCs exhibit very few SNAs and SVs, but are characterized by recurrent chromosome-wide copy number aberrations. Various RNA fusion events altering the MAP kinase pathway have been identified, suggesting a key role for kinase pathways in the development and growth of these devastating brain tumors. Citation Format: Diana M. Merino, Yongjin Li, Xiaotu Ma, Jinghui Zhang, David Malkin, Richard J. Gilbertson. Sequencing approaches define the mutation and fusion landscape of choroid plexus carcinomas. [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 3280. doi:10.1158/1538-7445.AM2015-3280