Intratumor heterogeneity (ITH) drives neoplastic progression and therapeutic resistance. We used the bioinformatics tools 'expanding ploidy and allele frequency on nested subpopulations' (EXPANDS) ...and PyClone to detect clones that are present at a ≥10% frequency in 1,165 exome sequences from tumors in The Cancer Genome Atlas. 86% of tumors across 12 cancer types had at least two clones. ITH in the morphology of nuclei was associated with genetic ITH (Spearman's correlation coefficient, ρ = 0.24-0.41; P < 0.001). Mutation of a driver gene that typically appears in smaller clones was a survival risk factor (hazard ratio (HR) = 2.15, 95% confidence interval (CI): 1.71-2.69). The risk of mortality also increased when >2 clones coexisted in the same tumor sample (HR = 1.49, 95% CI: 1.20-1.87). In two independent data sets, copy-number alterations affecting either <25% or >75% of a tumor's genome predicted reduced risk (HR = 0.15, 95% CI: 0.08-0.29). Mortality risk also declined when >4 clones coexisted in the sample, suggesting a trade-off between the costs and benefits of genomic instability. ITH and genomic instability thus have the potential to be useful measures that can universally be applied to all cancers.
Central nervous system (CNS) neoplasms are difficult to treat due to their sensitive location. Over the past two decades, the availability of patient tumor materials facilitated large scale genomic ...and epigenomic profiling studies, which have resulted in detailed insights into the molecular underpinnings of CNS tumorigenesis. Based on results from these studies, CNS tumors have high molecular and cellular intra-tumoral and inter-tumoral heterogeneity. CNS cancer models have yet to reflect the broad diversity of CNS tumors and patients and the lack of such faithful cancer models represents a major bottleneck to urgently needed innovations in CNS cancer treatment. Pediatric cancer model development is lagging behind adult tumor model development, which is why we focus this review on CNS tumors mutated for BRAF
V600E
which are more prevalent in the pediatric patient population. BRAF
V600E
-mutated CNS tumors exhibit high inter-tumoral heterogeneity, encompassing clinically and histopathological diverse tumor types. Moreover, BRAF
V600E
is the second most common alteration in pediatric low-grade CNS tumors, and low-grade tumors are notoriously difficult to recapitulate
in vitro
and
in vivo
. Although the mutation predominates in low-grade CNS tumors, when combined with other mutations, most commonly CDKN2A deletion, BRAF
V600E
-mutated CNS tumors are prone to develop high-grade features, and therefore BRAF
V600E
-mutated CNS are a paradigm for tumor progression. Here, we describe existing
in vitro
and
in vivo
models of BRAF
V600E
-mutated CNS tumors, including patient-derived cell lines, patient-derived xenografts, syngeneic models, and genetically engineered mouse models, along with their advantages and shortcomings. We discuss which research gaps each model might be best suited to answer, and identify those areas in model development that need to be strengthened further. We highlight areas of potential research focus that will lead to the heightened predictive capacity of preclinical studies, allow for appropriate validation, and ultimately improve the success of “bench to bedside” translational research.
Stem and progenitor cells are characterized by their ability to self-renew and produce differentiated progeny. A fine balance between these processes is achieved through controlled asymmetric ...divisions and is necessary to generate cellular diversity during development and to maintain adult tissue homeostasis. Disruption of this balance may result in premature depletion of the stem/progenitor cell pool, or abnormal growth. In many tissues, including the brain, dysregulated asymmetric divisions are associated with cancer. Whether there is a causal relationship between asymmetric cell division defects and cancer initiation is as yet not known. Here, we review the cellular and molecular mechanisms that regulate asymmetric cell divisions in the neural lineage and discuss the potential connections between this regulatory machinery and cancer.
Pediatric brainstem gliomas often harbor oncogenic K27M mutation of histone H3.3. Here we show that GSKJ4 pharmacologic inhibition of K27 demethylase JMJD3 increases cellular H3K27 methylation in ...K27M tumor cells and demonstrate potent antitumor activity both in vitro against K27M cells and in vivo against K27M xenografts. Our results demonstrate that increasing H3K27 methylation by inhibiting K27 demethylase is a valid therapeutic strategy for treating K27M-expressing brainstem glioma.
Glioblastoma multiforme (GBM) is an invariably fatal central nervous system tumor despite treatment with surgery, radiation, and chemotherapy. Further insights into the molecular and cellular ...mechanisms that drive GBM formation are required to improve patient outcome. MicroRNAs are emerging as important regulators of cellular differentiation and proliferation, and have been implicated in the etiology of a variety of cancers, yet the role of microRNAs in GBM remains poorly understood. In this study, we investigated the role of microRNAs in regulating the differentiation and proliferation of neural stem cells and glioblastoma-multiforme tumor cells.
We used quantitative RT-PCR to assess microRNA expression in high-grade astrocytomas and adult mouse neural stem cells. To assess the function of candidate microRNAs in high-grade astrocytomas, we transfected miR mimics to cultured-mouse neural stem cells, -mouse oligodendroglioma-derived stem cells, -human glioblastoma multiforme-derived stem cells and -glioblastoma multiforme cell lines. Cellular differentiation was assessed by immunostaining, and cellular proliferation was determined using fluorescence-activated cell sorting.
Our studies revealed that expression levels of microRNA-124 and microRNA-137 were significantly decreased in anaplastic astrocytomas (World Health Organization grade III) and glioblastoma multiforme (World Health Organization grade IV) relative to non-neoplastic brain tissue (P < 0.01), and were increased 8- to 20-fold during differentiation of cultured mouse neural stem cells following growth factor withdrawal. Expression of microRNA-137 was increased 3- to 12-fold in glioblastoma multiforme cell lines U87 and U251 following inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5-aza-dC). Transfection of microRNA-124 or microRNA-137 induced morphological changes and marker expressions consistent with neuronal differentiation in mouse neural stem cells, mouse oligodendroglioma-derived stem cells derived from S100 beta-v-erbB tumors and cluster of differentiation 133+ human glioblastoma multiforme-derived stem cells (SF6969). Transfection of microRNA-124 or microRNA-137 also induced G1 cell cycle arrest in U251 and SF6969 glioblastoma multiforme cells, which was associated with decreased expression of cyclin-dependent kinase 6 and phosphorylated retinoblastoma (pSer 807/811) proteins.
microRNA-124 and microRNA-137 induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme-derived stem cells and induce glioblastoma multiforme cell cycle arrest. These results suggest that targeted delivery of microRNA-124 and/or microRNA-137 to glioblastoma multiforme tumor cells may be therapeutically efficacious for the treatment of this disease.
This clinical trial evaluated whether whole exome sequencing (WES) and RNA sequencing (RNAseq) of paired normal and tumor tissues could be incorporated into a personalized treatment plan for newly ...diagnosed patients (<25 years of age) with diffuse intrinsic pontine glioma (DIPG). Additionally, whole genome sequencing (WGS) was compared to WES to determine if WGS would further inform treatment decisions, and whether circulating tumor DNA (ctDNA) could detect the H3K27M mutation to allow assessment of therapy response. Patients were selected across three Pacific Pediatric Neuro‐Oncology Consortium member institutions between September 2014 and January 2016. WES and RNAseq were performed at diagnosis and recurrence when possible in a CLIA‐certified laboratory. Patient‐derived cell line development was attempted for each subject. Collection of blood for ctDNA was done prior to treatment and with each MRI. A specialized tumor board generated a treatment recommendation including up to four FDA‐approved agents based upon the genomic alterations detected. A treatment plan was successfully issued within 21 business days from tissue collection for all 15 subjects, with 14 of the 15 subjects fulfilling the feasibility criteria. WGS results did not significantly deviate from WES‐based therapy recommendations; however, WGS data provided further insight into tumor evolution and fidelity of patient‐derived cell models. Detection of the H3F3A or HIST1H3B K27M (H3K27M) mutation using ctDNA was successful in 92% of H3K27M mutant cases. A personalized treatment recommendation for DIPG can be rendered within a multicenter setting using comprehensive next‐generation sequencing technology in a clinically relevant timeframe.
What's new?
While children diagnosed with diffuse intrinsic pontine glioma (DIPG) continue to suffer dismal survival outcomes, progress in next‐generation sequencing technologies have advanced the possibility of personalized therapeutic interventions. This prospective study demonstrates the feasibility of performing biopsies on patients with DIPG at diagnosis, applying approaches in next‐generation sequencing to determine an individualized therapy plan in a clinically relevant timeframe. Analyses of cell lines derived from patient samples revealed key genomic alterations typical of DIPG, including mutations in ACVR1, H3F3A/HIST1H3B, PIK3R1, PPM1D, and TP53. The study further highlights the utility of circulating tumor DNA for detecting driver mutations in DIPG.
Several cancer types consist of multiple genetically and phenotypically distinct subpopulations. The underlying mechanism for this intra-tumoral heterogeneity can be explained by the clonal evolution ...model, whereby growth advantageous mutations cause the expansion of cancer cell subclones. The recurrent phenotype of many cancers may be a consequence of these coexisting subpopulations responding unequally to therapies. Methods to computationally infer tumor evolution and subpopulation diversity are emerging and they hold the promise to improve the understanding of genetic and molecular determinants of recurrence.
To address cellular subpopulation dynamics within human tumors, we developed a bioinformatic method, EXPANDS. It estimates the proportion of cells harboring specific mutations in a tumor. By modeling cellular frequencies as probability distributions, EXPANDS predicts mutations that accumulate in a cell before its clonal expansion. We assessed the performance of EXPANDS on one whole genome sequenced breast cancer and performed SP analyses on 118 glioblastoma multiforme samples obtained from TCGA. Our results inform about the extent of subclonal diversity in primary glioblastoma, subpopulation dynamics during recurrence and provide a set of candidate genes mutated in the most well-adapted subpopulations. In summary, EXPANDS predicts tumor purity and subclonal composition from sequencing data.
EXPANDS is available for download at http://code.google.com/p/expands (matlab version--used in this manuscript) and http://cran.r-project.org/web/packages/expands (R version).
Oligodendrocyte progenitor cells (OPC) undergo asymmetric cell division (ACD) to generate one OPC and one differentiating oligodendrocyte (OL) progeny. Loss of pro-mitotic proteoglycan and OPC marker ...NG2 in the OL progeny is the earliest immunophenotypic change of unknown mechanism that indicates differentiation commitment. Here, we report that expression of the mouse homolog of Drosophila tumor suppressor Lethal giant larvae 1 (Lgl1) is induced during OL differentiation. Lgl1 conditional knockout OPC progeny retain NG2 and show reduced OL differentiation, while undergoing more symmetric self-renewing divisions at the expense of asymmetric divisions. Moreover, Lgl1 and hemizygous Ink4a/Arf knockouts in OPC synergistically induce gliomagenesis. Time lapse and total internal reflection microscopy reveals a critical role for Lgl1 in NG2 endocytic routing and links aberrant NG2 recycling to failed differentiation. These data establish Lgl1 as a suppressor of gliomagenesis and positive regulator of asymmetric division and differentiation in the healthy and demyelinated murine brain.
Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration ...determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin β1 and inhibits its downstream signaling, and Integrin β1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin β1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.
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•Lgl1 promotes mammary gland branching morphogenesis•Lgl1 is required for collective migration but not epithelial polarity•LGL1 binds to Integrin β1 and inhibits its downstream signaling•Branching mechanisms are distinct between mammary gland and invertebrate epithelia
Ma et al. show that Lgl1 is essential for mammary gland branching morphogenesis but not epithelial polarity. Lgl1 is required for directional migration by regulating Integrin β1 signaling levels and focal adhesion strengths. Finally, branching mechanisms are distinct between mammary gland and Drosophila systems where directional migration is indispensable.
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