TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6% ± 8.7%, respectively (p < 0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89% ± 2% vs. 57.4% ± 1.8% (p < 0.01)). In contrast, β-catenin mutation sensitized TP53 mutant cells to radiation (p < 0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5% ± 1.5% in lithium treated cells vs. 56.6 ± 3% (p < 0.01)) accompanied by increased number of γH2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33% ± 8% for lithium treated cells vs. 27% ± 3% for untreated controls (p = 0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.
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The online version of this article (doi:10.1186/s40478-014-0174-y) contains supplementary material, which is available to authorized users.
Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.
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The online version of this article (doi:10.1007/s00401-013-1198-2) contains supplementary material, which is available to authorized users.
TERT promoter mutations; SHH pathway; Adult; Medulloblastoma
Li-Fraumeni syndrome (LFS) is a rare dominantly inherited cancer predisposition syndrome that was first described in 1969. In most families, it is caused by germline mutations in the TP53 gene and is characterized by early onset of multiple specific cancers and very high lifetime cumulative cancer risk. Despite significant progress in understanding the molecular biology of TP53, the optimal clinical management of this syndrome is poorly defined. We convened a workshop on November 2, 2010, at the National Institutes of Health in Bethesda, Maryland, bringing together clinicians and scientists, as well as individuals from families with LFS, to review the state of the science, address clinical management issues, stimulate collaborative research, and engage the LFS family community. This workshop also led to the creation of the Li-Fraumeni Exploration (LiFE) Research Consortium.
Li-Fraumeni syndrome; hereditary cancer predisposition syndrome; TP53 mutations
Medulloblastoma, the most common malignant pediatric brain tumour, is currently treated with non-specific cytotoxic therapies including surgery, whole brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, prior attempts to identify targets for therapy have been underpowered due to small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup enriched. The most common region of focal copy number gain is a tandem duplication of the Parkinson’s disease gene SNCAIP, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1 that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGFβ signaling in Group 3, and NF-κB signaling in Group 4 suggest future avenues for rational, targeted therapy.
Adrenocortical carcinoma (ACC) is recognized to be a component tumor of the Li Fraumeni Syndrome (LFS), a familial cancer predisposition resulting from germline mutations in the p53 tumor-suppressor. p53 activity is tightly regulated by multiple post-translational mechanisms, disruption of which may lead to tumorigenesis. ACC is present in disproportionately high rates among p53-mutation carriers, suggesting tissue-specific manifestations of p53 deficiency. Additionally, p53-associated ACC demonstrates a strong predominance in infants and children. Several of the p53 alleles associated with pediatric ACC, however, retain significant wild-type activity and demonstrate incomplete penetrance, a finding distinct from other LFS-component tumors. In this review, we discuss the relationship between p53 and adrenocortical carcinogenesis, with specific focus on disease-specific alleles, tumorigenesis in the context of adrenal development and potential therapeutic approaches to p53-associated ACC.
Genomic rearrangements are thought to occur progressively during tumor development. Recent findings, however, suggest an alternative mechanism, involving massive chromosome rearrangements in a one-step catastrophic event termed chromothripsis. We report the whole-genome sequencing-based analysis of a Sonic-Hedgehog medulloblastoma (SHH-MB) brain tumor from a patient with a germline TP53 mutation (Li-Fraumeni syndrome), uncovering massive, complex chromosome rearrangements. Integrating TP53 status with microarray and deep sequencing-based DNA rearrangement data in additional patients reveals a striking association between TP53 mutation and chromothripsis in SHH-MBs. Analysis of additional tumor entities substantiates a link between TP53 mutation and chromothripsis, and indicates a context-specific role for p53 in catastrophic DNA rearrangements. Among these, we observed a strong association between somatic TP53 mutations and chromothripsis in acute myeloid leukemia. These findings connect p53 status and chromothripsis in specific tumor types, providing a genetic basis for understanding particularly aggressive subtypes of cancer.
Purpose: To identify and examine polymorphisms of genes associated with aggressive and clinical significant forms of prostate cancer among a screening cohort.
Experimental Design: We conducted a genome-wide association study among patients with aggressive forms of prostate cancer and biopsy-proven normal controls ascertained from a prostate cancer screening program. We then examined significant associations of specific polymorphisms among a prostate cancer screened cohort to examine their predictive ability in detecting prostate cancer.
Results: We found significant associations between aggressive prostate cancer and five single nucleotide polymorphisms (SNPs) in the 10q26 (rs10788165, rs10749408, and rs10788165, p value for association 1.3 × 10−10 to 3.2 × 10−11) and 15q21 (rs4775302 and rs1994198, p values for association 3.1 × 10−8 to 8.2 × 10−9) regions. Results of a replication study done in 3439 patients undergoing a prostate biopsy, revealed certain combinations of these SNPs to be significantly associated not only with prostate cancer but with aggressive forms of prostate cancer using an established classification criterion for prostate cancer progression (odds ratios for intermediate to high-risk disease 1.8–3.0, p value 0.003–0.001). These SNP combinations were also important clinical predictors for prostate cancer detection based on nomogram analysis that assesses prostate cancer risk.
Conclusions: Five SNPs were found to be associated with aggressive forms of prostate cancer. We demonstrated potential clinical applications of these associations.
Osteosarcoma (OS) is a rare malignant bone tumor with an overall incidence rate of 4.6 cases per million children aged 0-19 years in the United States. While the etiology of OS is largely unknown, its distinctive age-incidence pattern suggests that growth and development is crucial in genesis. Prior studies have suggested that variants in genes in the estrogen metabolism (ESTR) and insulin-like growth factor/growth hormone (IGF/GH) pathways are associated with OS. We examined 798 single nucleotide polymorphisms (SNPs) in 42 genes from these pathways in a case-parent study (229 complete triads and 56 dyads) using buccal cell samples. Relative risks (RR) and 95% confidence intervals (CI) associated with transmitting one or two copies of the variant were estimated using log-linear models. After Bonferroni correction, 1 SNP within the ESTR pathway (rs1415270: RR = 0.50 and 8.37 for 1 and 2 vs. 0 copies, respectively; p = 0.010), and two SNPs in the IGF/GH pathway (rs1003737: RR = 0.91 and 0.0001 for 1 and 2 vs. 0 copies, respectively; p <0.0001 and rs2575352: RR = 2.62 and 0.22 for 1 and 2 vs. 0 copies; p < 0.0001) were significantly associated with OS incidence. These results confirm previous findings that variation in the estrogen metabolism and bone growth pathways influence OS risk and further support a biologically and epidemiologically plausible role in OS development.
Osteosarcoma; case-parent study; growth and development; insulin-like growth factor pathway; estrogen metabolism pathway
The diagnosis of medulloblastoma likely encompasses several distinct entities, with recent evidence for the existence of at least four unique molecular subgroups that exhibit distinct genetic, transcriptional, demographic, and clinical features. Assignment of molecular subgroup through routine profiling of high-quality RNA on expression microarrays is likely impractical in the clinical setting. The planning and execution of medulloblastoma clinical trials that stratify by subgroup, or which are targeted to a specific subgroup requires technologies that can be economically, rapidly, reliably, and reproducibly applied to formalin-fixed paraffin embedded (FFPE) specimens. In the current study, we have developed an assay that accurately measures the expression level of 22 medulloblastoma subgroup-specific signature genes (CodeSet) using nanoString nCounter Technology. Comparison of the nanoString assay with Affymetrix expression array data on a training series of 101 medulloblastomas of known subgroup demonstrated a high concordance (Pearson correlation r = 0.86). The assay was validated on a second set of 130 non-overlapping medulloblastomas of known subgroup, correctly assigning 98% (127/130) of tumors to the appropriate subgroup. Reproducibility was demonstrated by repeating the assay in three independent laboratories in Canada, the United States, and Switzerland. Finally, the nanoString assay could confidently predict subgroup in 88% of recent FFPE cases, of which 100% had accurate subgroup assignment. We present an assay based on nanoString technology that is capable of rapidly, reliably, and reproducibly assigning clinical FFPE medulloblastoma samples to their molecular subgroup, and which is highly suited for future medulloblastoma clinical trials.
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The online version of this article (doi:10.1007/s00401-011-0899-7) contains supplementary material, which is available to authorized users.
Medulloblastoma; Molecular classification; Clinical trials; NanoString
High-throughput genotyping data are useful for making inferences about human evolutionary history. However, the populations sampled to date are unevenly distributed, and some areas (e.g., South and Central Asia) have rarely been sampled in large-scale studies. To assess human genetic variation more evenly, we sampled 296 individuals from 13 worldwide populations that are not covered by previous studies. By combining these samples with a data set from our laboratory and the HapMap II samples, we assembled a final dataset of ~250,000 SNPs in 850 individuals from 40 populations. With more uniform sampling, the estimate of global genetic differentiation (FST) substantially decreases from ~16% with the HapMap II samples to ~11%. A panel of copy number variations typed in the same populations shows patterns of diversity similar to the SNP data, with highest diversity in African populations. This unique sample collection also permits new inferences about human evolutionary history. The comparison of haplotype variation among populations supports a single out-of-Africa migration event and suggests that the founding population of Eurasia may have been relatively large but isolated from Africans for a period of time. We also found a substantial affinity between populations from central Asia (Kyrgyzstani and Mongolian Buryat) and America, suggesting a central Asian contribution to New World founder populations.
Single nucleotide polymorphism array; SNP; population structure; population diversity; human population history
Li-Fraumeni syndrome (LFS) is a classic cancer predisposition disorder that is commonly associated with germline mutations of the p53 tumor suppressor gene. Examination of the wide spectrum of adult-onset and childhood cancers and the distribution of p53 mutations has led to a greater understanding of cancer genotype-phenotype correlations. However, the complex LFS phenotype is not readily explained by the simple identification of germline p53 mutations in affected individuals. Recent work has identified genetic events that modify the LFS phenotype. These include intragenic polymorphisms, mutations/polymorphisms of genes in the p53 regulatory pathway, as well as more global events such as aberrant copy number variation and telomere attrition. These genetic events may, in part, explain the breadth of tumor histiotypes within and across LFS families, the apparent accelerated age of onset within families, and the range of clinical outcomes among affected family members. This review will examine the clinical and genetic definitions of LFS and offer insight into how lessons learned from the study of this rare disorder may inform similar questions in other familial cancer syndromes.
Li-Fraumeni syndrome; cancer predisposition; germline p53 mutations
DNA copy number variations (CNVs) are an important component of genetic variation, affecting a greater fraction of the genome than single nucleotide polymorphisms (SNPs). The advent of high-resolution SNP arrays has made it possible to identify CNVs. Characterization of widespread constitutional (germline) CNVs has provided insight into their role in susceptibility to a wide spectrum of diseases, and somatic CNVs can be used to identify regions of the genome involved in disease phenotypes. The role of CNVs as risk factors for cancer is currently underappreciated. However, the genomic instability and structural dynamism that characterize cancer cells would seem to make this form of genetic variation particularly intriguing to study in cancer. Here, we provide a detailed overview of the current understanding of the CNVs that arise in the human genome and explore the emerging literature that reveals associations of both constitutional and somatic CNVs with a wide variety of human cancers.
Although most reports describe the protein kinase integrin-linked kinase (ILK) as a proto-oncogene, occasional studies detail opposing functions in the regulation of normal and transformed cell proliferation, differentiation, and apoptosis. Here, we demonstrated that ILK functions as an oncogene in the highly aggressive pediatric sarcoma alveolar rhabdomyosarcoma (ARMS) and as a tumor suppressor in the related embryonal rhabdomyosarcoma (ERMS). These opposing functions hinge on signaling through a noncanonical ILK target, JNK1, to the proto-oncogene c-Jun. RNAi-mediated depletion of ILK induced activation of JNK and its target, c-Jun, resulting in growth of ERMS cells, whereas in ARMS cells, it led to loss of JNK/c-Jun signaling and suppression of growth both in vitro and in vivo. Ectopic expression of the fusion gene characteristic of ARMS (paired box 3–forkhead homolog in rhabdomyosarcoma [PAX3-FKHR]) in ERMS cells was sufficient to convert them to an ARMS signaling phenotype and render ILK activity oncogenic. Furthermore, restoration of JNK1 in ARMS reestablished a tumor-suppressive function for ILK. These findings indicate what we believe to be a novel effector pathway regulated by ILK, provide a mechanism for interconversion of oncogenic and tumor-suppressor functions of a single regulatory protein based on the genetic background of the tumor cells, and suggest a rationale for tailored therapy of rhabdomyosarcoma based on the different activities of ILK.
The genome era in medicine is upon us. Questions that arise from patient and family care are a watershed for research and technology, which in turn fuel the cycle of opportunity for impact through delivery of health services, which feeds back to families. Medical infrastructure needs to adapt to the dramatic pace of technology development in the wake of the Human Genome Project, in order for genome data to be delivered as information and applied as knowledge to benefit health.
In contrast to the classical view of development as a preprogrammed and deterministic process, recent studies have demonstrated that stochastic perturbations of highly non-linear systems may underlie the emergence and stability of biological patterns. Herein, we address the question of whether noise contributes to the generation of the stereotypical temporal pattern in gene expression during flower development. We modeled the regulatory network of organ identity genes in the Arabidopsis thaliana flower as a stochastic system. This network has previously been shown to converge to ten fixed-point attractors, each with gene expression arrays that characterize inflorescence cells and primordial cells of sepals, petals, stamens, and carpels. The network used is binary, and the logical rules that govern its dynamics are grounded in experimental evidence. We introduced different levels of uncertainty in the updating rules of the network. Interestingly, for a level of noise of around 0.5–10%, the system exhibited a sequence of transitions among attractors that mimics the sequence of gene activation configurations observed in real flowers. We also implemented the gene regulatory network as a continuous system using the Glass model of differential equations, that can be considered as a first approximation of kinetic-reaction equations, but which are not necessarily equivalent to the Boolean model. Interestingly, the Glass dynamics recover a temporal sequence of attractors, that is qualitatively similar, although not identical, to that obtained using the Boolean model. Thus, time ordering in the emergence of cell-fate patterns is not an artifact of synchronous updating in the Boolean model. Therefore, our model provides a novel explanation for the emergence and robustness of the ubiquitous temporal pattern of floral organ specification. It also constitutes a new approach to understanding morphogenesis, providing predictions on the population dynamics of cells with different genetic configurations during development.
Spontaneous tumor regression is a unique feature of pediatric low-grade gliomas (PLGG). We speculated that lack of telomere maintenance is responsible for this behavior. We first looked for evidence of telomerase activity and alternative-lengthening telomeres (ALT) in 56 PLGG. Telomerase activity was observed in 0 of 11 PLGG in contrast to 10 of 13 high-grade pediatric brain tumors. There was no ALT in 45 of 45 samples. We applied Q-FISH to eight patients whose indolent PLGG underwent two metachronous biopsies over a lag of several years. Telomere shortening was observed in the second biopsy in all tumors but not in a normal brain control (P < .0001), indicating that lack of telomere maintenance is associated with continuous telomere erosion. Based on these observations, we observed that younger PLGG patients who exhibit more aggressive and frequently recurrent tumors had significantly longer telomeres than older ones (P = .00014). Tumors with a terminal restriction fragment length of <7.5 did not recur, whereas the presence of longer telomeres (>8.0) conferred a high likelihood of late recurrences in PLGG. Our findings provide a plausible biological mechanism to explain the tendency of PLGG to exhibit growth arrest and spontaneous regression. Telomere maintenance may therefore represent the first known biologic prognostic marker in PLGG.
Telomere; telomerase; senescence; low-grade glioma; prognosis
The purpose of this study was to explore the attitudes of genomics researchers in a pediatric setting in the context of regulatory guidance recommending the disclosure of clinically significant research findings.
A validated 32-item questionnaire was sent to 107 researchers with two large-scale projects (the Canadian Pediatric Cancer Genome Consortium and the Finding of Rare Genes Canada Consortium). We examined researchers' attitudes toward obligations to offer genomic research results (including if the participant was deceased, a relative, or a child), influence of the certainty/severity of the condition on this obligation, and personal experiences.
Of the 107 researchers, 74 (69%) responded. Researchers did not feel a strong responsibility to look for meaningful incidental results in the research genomic data set (n = 27, 37%). However, once identified, they felt participants had a strong right to receive them, irrespective of being incidental (n = 50, 68%) or primary targets (n = 64, 87%). There was a high degree of support for informing siblings of genomic results (n = 46, 62%), especially for treatable conditions (n = 56, 76%). Less than half of the participants indicated that their research ethics board required an offer of results (n = 34, 46%) or provided a detailed process (n = 16, 22%).
Researchers strongly support the offer of targeted and incidental genomic research results to participants. Greater regulatory guidance is needed for a consistent approach.
genomics; incidental findings; next-generation sequencing; researchers
Cancer evolves by mutation, with somatic reactivation of retrotransposons being one such mutational process. Germline retrotransposition can cause processed pseudogenes, but whether this occurs somatically has not been evaluated. Here we screen sequencing data from 660 cancer samples for somatically acquired pseudogenes. We find 42 events in 17 samples, especially non-small cell lung cancer (5/27) and colorectal cancer (2/11). Genomic features mirror those of germline LINE element retrotranspositions, with frequent target-site duplications (67%), consensus TTTTAA sites at insertion points, inverted rearrangements (21%), 5′ truncation (74%) and polyA tails (88%). Transcriptional consequences include expression of pseudogenes from UTRs or introns of target genes. In addition, a somatic pseudogene that integrated into the promoter and first exon of the tumour suppressor gene, MGA, abrogated expression from that allele. Thus, formation of processed pseudogenes represents a new class of mutation occurring during cancer development, with potentially diverse functional consequences depending on genomic context.
Germline pseudogenes have an important role in human evolution. Here, the authors analyse sequencing data from 660 cancer samples and find evidence for the formation of somatically acquired pseudogenes, a new class of mutation, which may contribute to cancer development.
Squalene, an isoprenoid antioxidant is a potential cytoprotective agent against chemotherapy-induced toxicity. We have previously published that squalene protects light-density bone marrow cells against cis-diamminedichloroplatinum( II) (cisplatin)-induced toxicity without protecting tumor cells in vitro. Here, we developed an in vivo mouse model of cisplatin and cis-diammine (cyclobutane-1,1-dicarboxylato) platinum(II) (carboplatin)-induced toxicity to further investigate squalene-mediated LD-BM cytoprotection including the molecular mechanism behind selective cytoprotection. We found that squalene significantly reduced the body weight loss of cisplatin and carboplatin-treated mice. Light-density bone marrow cells from squalene-treated mice exhibited improved formation of hematopoietic colonies (colony-forming unit-granulocyte macrophage). Furthermore, squalene also protected mesenchymal stem cell colonies (colony-forming unit-fibroblast) from cisplatin and carboplatin-induced toxicity. Squalene-induced protection was associated with decreased reactive oxygen species and increased levels of glutathione and glutathione peroxidase/glutathione-S-transferase. Importantly, squalene did not protect neuroblastoma, small cell carcinoma, or medulloblastoma xenografts against cisplatin-induced toxicity. These results suggest that squalene is a potential candidate for future development as a cytoprotective agent against chemotherapeutic toxicity.