Search

Background Chemotherapy improves overall survival after surgery and radiotherapy for newly diagnosed high-risk IDH-mutant low-grade gliomas (LGGs), but a proportion of patients treated with temozolomide (TMZ) will develop recurrent tumors with TMZ-induced hypermutation. We aimed to determine the prevalence of TMZ-induced hypermutation at recurrence and prognostic implications. Methods We sequenced recurrent tumors from 82 patients with initially low-grade IDH-mutant gliomas who underwent reoperation and correlated hypermutation status with grade at recurrence and subsequent clinical outcomes. Results Hypermutation was associated with high-grade disease at the time of reoperation (OR 12.0 95% CI 2.5-115.5, P = .002) and was identified at transformation in 57% of recurrent LGGs previously exposed to TMZ. After anaplastic (grade III) transformation, hypermutation was associated with shorter survival on univariate and multivariate analysis (HR 3.4, 95% CI 1.2-9.9, P = .024), controlling for tumor grade, subtype, age, and prior radiotherapy. The effect of hypermutation on survival after transformation was validated in an independent, published dataset. Hypermutated (HM) tumors were more likely to develop discontiguous foci of disease in the brain and spine (P = .003). To estimate the overall incidence of high-grade transformation among low-grade IDH-mutant tumors, data from a phase II trial of TMZ for LGG were analyzed. Eight-year transformation-free survival was 53.8% (95% CI 42.8-69.2), and 61% of analyzed transformed cases were HM. Conclusions TMZ-induced hypermutation is a common event in transformed LGG previously treated with TMZ and is associated with worse prognosis and development of discontiguous disease after recurrence. These findings impact tumor classification at recurrence, prognostication, and clinical trial design.

Tumor cell immortality is a fundamental hallmark of human cancers. Normally silenced during somatic cell differentiation, 90% of human tumors reactivate Telomerase Reverse Transcriptase (TERT) expression to achieve cellular immortality. TERT, the catalytic subunit of telomerase, complexes with the RNA template molecule TERC to maintain telomeres. Mutations in the TERT promoter (TERTp) are the most common non-coding mutation across all cancer types and the most frequent mutation within many cancers, such as IDH wildtype glioblastoma (GBM), Melanoma, and Bladder Cancer. TERTp mutations generate de novo E26 Transformation Specific (ETS) binding motifs that are spaced full helical turns from TERTp native ETS sites. Together the de novo and native ETS motifs specifically recruit the GABP tetrameric complex but not the GABP dimer. CRISPR-cas9 mediated insertion/deletion mutagenesis of the unique exon of GABP tetramer forming subunit, GABPB1L (B1L), reduces TERT transcriptional activity in a TERT promoter-mutation dependent manner. Here we show that GABPB1S (B1S), the GABP dimer restricted alternative isoform of GABPB1, is consistently and significantly increased following B1L reduction, a process we have determined to be driven by a conserved homeostatic mechanism whereby the GABP tetramer suppresses expression of one of its own components, GABPB1. In contrast to the native setting, in the absence of B1L the elevated B1S expression leads to dimer binding to the mutant TERTp and maintenance of TERT expression. Indeed, co-targeting B1L and B1S together, but not B1L alone, via CRISPR-cas9 knockout resulted in a near complete elimination of GABP recruitment to the TERTp and TERT expression, and lead to tumor cell death and eventual senescence in a telomere length dependent manner. Together, this data suggests a new model of the TERT-GABP axis involving the tetramer and dimer and highlights a new and potentially more potent therapeutic strategy to eliminate TERT expression and reverse tumor cell immortality.

The prospect of using immunotherapy for IDH-mutant LGGs has been daunting given the immune-poor microenvironment and low mutational burden. We hypothesized that LGG-targeting T cells might still be present at low frequency and with limited regional infiltration into the tumor. To improve sensitivity, we combined high-density multi-region tumor sampling with high-throughput neoantigen-T cell screening for a patient with WHO Grade II diffuse astrocytoma who eventually progressed, at second recurrence, to anaplastic astrocytoma. We performed maximal-anatomic sampling from 10 distinct regions of the tumor at the initial resection, as well as single sampling at first recurrence, for exome-based prediction of clonal and subclonal expressed neoantigens, RNAseq-based estimation of regional immune cell composition, and T cell receptor (TCR) beta deep sequencing. Based on our predictions, we then generated a barcoded library of patient-specific peptide-HLA multimers loaded with predicted neopeptides. Using this library, neoantigen-specific CD8 T cells were captured and isolated from patient peripheral blood and subjected to single cell TCR sequencing. We screened patient-derived peripheral blood drawn two years after initial resection and identified five T cell clones recognizing three LGG neoepitopes. Two neoepitopes were derived from truncal, tumor-wide mutations, including a truncating splice-site mutation in TP53 and a missense mutation in MRPL46. Each of these neoepitopes were recognized by two distinct TCRs, consistent with TCR convergence. A third neoepitope was derived from a subclonal MRPL46 mutation. Using the TCR beta sequence as a molecular barcode, TCRs specific to the 2 clonal neoepitopes, but not the subclonal neoepitope, were detectable in the glioma and blood at initial resection, as well as in the recurrent glioma. In summary, we demonstrate the existence and persistence of neoantigen-targeting T cells within the blood and tumor of an IDH-mutant LGG patient. These findings suggest a feasible methodology to develop personalized T cell-based immunotherapies for IDH-mutant LGGs.

Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and generation of therapy-resistant clones. While ITH in glioblastoma has been well-characterized at the genomic and transcriptomic levels, the extent of ITH at the epigenomic level and its biological and clinical significance are not well understood. In collaboration with neurosurgeons, neuropathologists, and biomedical imaging experts, we have established a novel topographical approach towards characterizing epigenomic ITH in three-dimensional (3-D) space. We utilize pre-operative MRI scans to define tumor volume and then utilize 3-D surgical neuro-navigation to intra-operatively acquire 10+ samples representing maximal anatomical diversity. The precise spatial location of each sample is mapped by 3-D coordinates, enabling tumors to be visualized in 360-degrees and providing unprecedented insight into their spatial organization and patterning. For each sample, we conduct assay for transposase-accessible chromatin using sequencing (ATAC-Seq), which provides information on the genomic locations of open chromatin, DNA-binding proteins, and individual nucleosomes at nucleotide resolution. We additionally conduct whole-exome sequencing and RNA sequencing for each spatially mapped sample. Integrative analysis of these datasets reveals distinct patterns of chromatin accessibility within glioblastoma tumors, as well as their associations with genetically defined clonal expansions. Our analysis further reveals how differences in chromatin accessibility within tumors reflect underlying transcription factor activity at gene regulatory elements, including both promoters and enhancers, and drive expression of particular gene expression sets, including neuronal and immune programs. Collectively, this work provides the most comprehensive characterization of epigenomic ITH to date, establishing its importance for driving tumor evolution and therapy resistance in glioblastoma. As a resource for further investigation, we have provided our datasets on an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

Aim: To provide a comprehensive understanding of gene regulatory networks in the developing human brain and a foundation for interpreting pathogenic deregulation. Materials & methods: We generated reference epigenomes and transcriptomes of dissected brain regions and primary neural progenitor cells (NPCs) derived from cortical and ganglionic eminence tissues of four normal human fetuses. Results: Integration of these data across developmental stages revealed a directional increase in active regulatory states, transcription factor activities and gene transcription with developmental stage. Consistent with differences in their biology, NPCs derived from cortical and ganglionic eminence regions contained common, region specific, and gestational week specific regulatory states. Conclusion: We provide a high-resolution regulatory network for NPCs from different brain regions as a comprehensive reference for future studies.

Background Emerging data suggest that a subset of patients with diffuse isocitrate dehydrogenase (IDH)-mutant low-grade glioma (LGG) who receive adjuvant temozolomide (TMZ) recur with hypermutation in association with malignant progression to higher-grade tumors. It is currently unclear why some TMZ-treated LGG patients recur with hypermutation while others do not. MGMT encodes O6-methylguanine-DNA methyltransferase, a DNA repair protein that removes cytotoxic and potentially mutagenic lesions induced by TMZ. Here, we hypothesize that epigenetic silencing of MGMT by promoter methylation facilitates TMZ-induced mutagenesis in LGG patients and contributes to development of hypermutation at recurrence. Methods We utilize a quantitative deep sequencing assay to characterize MGMT promoter methylation in 109 surgical tissue specimens from initial tumors and post-treatment recurrences of 37 TMZ-treated LGG patients. We utilize methylation arrays to validate our sequencing assay, RNA sequencing to assess the relationship between methylation and gene expression, and exome sequencing to determine hypermutation status. Results Methylation level at the MGMT promoter is significantly higher in initial tumors of patients that develop hypermutation at recurrence relative to initial tumors of patients that do not (45.7% vs 34.8%, P = 0.027). Methylation level in initial tumors can predict hypermutation at recurrence in univariate models and multivariate models that incorporate patient age and molecular subtype. Conclusions These findings reveal a mechanistic basis for observed differences in patient susceptibility to TMZ-driven hypermutation. Furthermore, they establish MGMT promoter methylation level as a potential biomarker to inform clinical management of LGG patients, including monitoring and treatment decisions, by predicting risk of hypermutation at recurrence.

TERT promoter mutation (TPM), found in over 80% of IDH-wildtype glioblastomas (GBMs) and oligodendrogliomas, leads to reactivation of telomerase and consequently tumor cell immortalization, which is potentially reversible. TERT could therefore serve as an effective target in treating tumors with TPM, if TPM is present throughout the tumor. Previous studies using a single sample or minimal sampling per tumor have shown potentially conflicting results, suggesting TPM is clonal in some cases and subclonal in others. Here we use spatially mapped tumor samples representing maximal tumor sampling to address this critical issue. Sanger sequencing was performed on 311 newly diagnosed and recurrent tumor samples from 19 IDH-wildtype GBMs and 10 oligodendrogliomas. To validate Sanger sequencing and resolve potentially ambiguous samples, deep amplicon sequencing was performed on 164 samples. To determine tumor purity and TERT expression levels, whole exome sequencing (164 samples) and RNA-Seq (129 samples) data sets were analyzed computationally. Sanger and amplicon sequencing showed that TPM was present in 305 of 311 samples (98.1%). TPM was not detected in 6 samples which had tumor purity estimates too low to be accurately determined by FACETS and lacked evidence of any driver mutation. Variant allele frequencies (VAFs) of TPM showed high positive correlation with those of clonal alterations in GBMs (r(90) = .93, p < .0001) and oligodendrogliomas (r(48) = .96, p < .0001). TPM VAFs also showed high positive correlation with tumor purity in both GBMs (r(112) = .92, p < .0001) and oligodendrogliomas (r(48) = .89, p < .0001). TPM VAF showed a moderate positive correlation with TERT expression in GBMs (r(78) = .40, p < .001) and oligodendrogliomas (r(47) = .49, p < .001). Therefore, TPM is a tumor-wide, clonal mutation in both newly diagnosed and recurrent GBMs and oligodendrogliomas. TPM VAF is moderately correlated with TERT expression.

Treatment failure in glioblastoma is often attributed to intratumoral heterogeneity (ITH), which fosters tumor evolution and selection of therapy-resistant clones. While genomic alterations are known contributors to ITH, emerging studies highlight functional roles for epigenomic ITH which integrates differentiation status, stochastic events, and microenvironmental inputs. Here, we have established a novel platform for integrative characterization of genomic and epigenomic ITH of glioblastoma in three-dimensional (3-D) space. In collaboration with neurosurgeons and biomedical imaging experts, we utilize 3-D surgical neuro-navigation to safely acquire ~10 tumor samples per patient representing maximal anatomical diversity. We conduct whole-exome sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-Seq) on each sample. The spatial location of each sample is mapped by its 3-D coordinates, allowing 360-degree visualization of genomic and epigenomic ITH for each patient. We demonstrate this approach on 8 patients with primary IDH-WT glioblastoma (83 spatially mapped samples), providing unprecedented insight into their spatial organization at the genomic and epigenomic levels. We link genetically defined tumor subclones to patterns of open chromatin and gene regulation, revealing underlying transcription factor binding at active promoters and enhancers. We also identify ITH in whole-genome doubling and focal oncogene amplification events in multiple patients, which we then link with epigenomic ITH. Further, to study microenvironmental inputs and their contribution to epigenomic ITH, we conduct deconvolution of RNA sequencing and ATAC-Seq data by analyzing feature co-variation. We resolve the 3-D spatial organization of immune, neural, and other nontumor cell types present in glioblastoma, characterizing their functional states and interactions with tumor cells. This work provides the most comprehensive spatial characterization of genomic and epigenomic ITH to date in glioblastoma. As a resource for further investigation, we have developed an interactive data sharing platform – The 3D Glioma Atlas – that enables 360-degree visualization of both genomic and epigenomic ITH.

The design and evaluation of immunotherapies in IDH-mutant lower grade gliomas (LGG) is hindered by a poor understanding of the LGG T-cell repertoire. We present data on the temporal evolution, intratumoral spatial distribution, and prognostic value of the T-cell repertoire in IDH-mutant LGGs. We performed immunogenomic profiling using T-cell receptor beta-chain sequencing of 163 glioma and peripheral blood samples from 33 immunotherapy-naive glioma patients (22 astrocytomas, 11 oligodendrogliomas). T-cell repertoire evolution was analyzed in a subset of 26 patients (69 samples) with matched primary (WHO grade II) and recurrent (WHO grade II-IV) glioma samples. T-cell repertoire diversity was defined as the number of unique T-cell clonotypes by V-gene, J-gene, and CDR3 nucleotide sequences. Malignant transformed (Grade III or IV) recurrent gliomas demonstrated increased T-cell repertoire diversity compared to their patient-matched primary tumors (p=0.0023), but grade II recurrences did not show the same increased diversity (p=0.26). This increase in T-cell repertoire diversity was greater in patients who underwent transformation in the context of TMZ-associated hypermutation compared to spontaneously transformed counterparts (p=0.035). In grade II primary astrocytomas (n=17), T-cell repertoire diversity above the median (186 unique T-cell clonotypes per sample) was associated with worse transformation-free (HR=4.2, p=0.045) and overall survival (HR=6.4, p=0.025). Next, we evaluated intratumoral immune heterogeneity in 7 patients by sampling from up to 10 distinct and maximally-separated intratumoral sites per LGG (64 samples). Eighty-two to 96% of unique clonotypes within a given tumor were present only within a single sampled site. Despite this heterogeneity, six LGG patients harbored T-cell clonotypes present tumor-wide across all sampled sites within a given tumor. Ten of 24 (42%) tumor-wide T-cell clonotypes were enriched in the glioma compared to matched peripheral blood, suggesting glioma-specificity. Taken together, T-cell receptor profiling in LGGs may have utility both as a prognostic biomarker and to identify glioma-specific T-cells.

The human placenta and its specialized cytotrophoblasts rapidly develop, have a compressed lifespan, govern pregnancy outcomes, and program the offspring's health. Understanding the molecular underpinnings of these behaviors informs development and disease. Profiling the extraembryonic epigenome and transcriptome during the 2nd and 3rd trimesters revealed H3K9 trimethylation overlapping deeply DNA hypomethylated domains with reduced gene expression and compartment-specific patterns that illuminated their functions. Cytotrophoblast DNA methylation increased, and several key histone modifications decreased across the genome as pregnancy advanced. Cytotrophoblasts from severe preeclampsia had substantially increased H3K27 acetylation globally and at genes that are normally downregulated at term but upregulated in this syndrome. In addition, some cases had an immature pattern of H3K27ac peaks, and others showed evidence of accelerated aging, suggesting subtype-specific alterations in severe preeclampsia. Thus, the cytotrophoblast epigenome dramatically reprograms during pregnancy, placental disease is associated with failures in this process, and H3K27 hyperacetylation is a feature of severe preeclampsia.

// Pavithra Viswanath 1 , Chloe Najac 1 , Jose L. Izquierdo-Garcia 1 , Aleksandr Pankov 2 , Chibo Hong 2 , Pia Eriksson 1 , Joseph F. Costello 2 , Russell O. Pieper 2 , Sabrina M. Ronen 1 1 Department of Radiology and Biomedical Imaging, University of California San Francisco, San Francisco, CA 94143, USA 2 Department of Neurological Surgery, Helen Diller Research Center, University of California San Francisco, San Francisco, CA 94143, USA Correspondence to: Sabrina Ronen, e-mail: sabrina.ronen@ucsf.edu Keywords: MCT1, MCT4, mutant IDH1, metabolic reprogramming, magnetic resonance spectroscopy Received: February 05, 2016 Accepted: April 10, 2016 Published: April 26, 2016 ABSTRACT Mutations in isocitrate dehydrogenase 1 (IDH1) are characteristic of low-grade gliomas. We recently showed that mutant IDH1 cells reprogram cellular metabolism by down-regulating pyruvate dehydrogenase (PDH) activity. Reduced pyruvate metabolism via PDH could lead to increased pyruvate conversion to lactate. The goal of this study was therefore to investigate the impact of the IDH1 mutation on the pyruvate-to-lactate flux. We used 13 C magnetic resonance spectroscopy and compared the conversion of hyperpolarized [1- 13 C]-pyruvate to [1- 13 C]-lactate in immortalized normal human astrocytes expressing mutant or wild-type IDH1 (NHAIDHmut and NHAIDHwt). Our results indicate that hyperpolarized lactate production is reduced in NHAIDHmut cells compared to NHAIDHwt. This reduction was associated with lower expression of the monocarboxylate transporters MCT1 and MCT4 in NHAIDHmut cells. Furthermore, hyperpolarized lactate production was comparable in lysates of NHAIDHmut and NHAIDHwt cells, wherein MCTs do not impact hyperpolarized pyruvate delivery and lactate production. Collectively, our findings indicated that lower MCT expression was a key contributor to lower hyperpolarized lactate production in NHAIDHmut cells. The SLC16A3 (MCT4) promoter but not SLC16A1 (MCT1) promoter was hypermethylated in NHAIDHmut cells, pointing to possibly different mechanisms mediating reduced MCT expression. Finally analysis of low-grade glioma patient biopsy data from The Cancer Genome Atlas revealed that MCT1 and MCT4 expression was significantly reduced in mutant IDH1 tumors compared to wild-type. Taken together, our study shows that reduced MCT expression is part of the metabolic reprogramming of mutant IDH1 gliomas. This finding could impact treatment and has important implications for metabolic imaging of mutant IDH1 gliomas.

2506 Background: Temozolomide, a commonly used alkylating agent to treat gliomas, can induce somatic hypermutation. The prevalence and clinical implications of this phenomenon are not well characterized. Methods: We used targeted and whole exome sequencing from a cohort of 82 patients with recurrent IDH-mutant low grade gliomas undergoing re-operation to evaluate the prevalence as well as the clinical implications of hypermutation. Results: Hypermutation was identified at transformation in 57% of recurrent gliomas exposed to Temozolomide, 94% of which were transformed to higher WHO grades. All patients who developed hypermutation were exposed to Temozolomide. Hypermutation was associated with transformation to higher WHO grade (OR 12.0 95% CI 2.5 – 115.5, p = 0.002) and shorter survival after transformation (HR 2.1, 95% CI 1.1-4.0, p = 0.018) compared with non-hypermutated transformed tumors, controlling for grade, molecular subtype, age, and prior radiotherapy. Patients with transformation to glioblastoma had poor survival regardless of hypermutation (p = 0.78). Hypermutated tumors were associated with development of discontiguous disease at a significantly higher frequency (p = 0.003), including four cases with spinal dissemination. Conclusions: TMZ-induced hypermutation is associated with high grade transformation, unique patterns of dissemination and shortened survival after transformation. Next generation sequencing should be considered in this patient population. These data have important implications for the management of newly diagnosed and recurrent IDH-mutant low grade gliomas.

BACKGROUND Previous studies of solid tumors have been restricted in their ability to map how heterogeneous cell populations evolved within the tumor in three-dimensional (3D) space due to insufficient sampling, typically one sample per tumor, and a lack of knowledge of where within the tumor the sample was obtained. Knowledge of the extensivity of heterogeneity and how it is spatially distributed is crucial for assessing whether a therapeutic target is truly tumor-wide, and for exploring how mutations relate to heterogeneity in the local microenvironment. METHODS We developed a novel platform to integrate and visualize in 3D multi-omics data generated from each of 8–10 spatially mapped samples per tumor. Together, the 171 samples collected using this approach from 16 adult diffuse glioma at diagnosis and recurrence form a novel resource – the 3D Glioma Atlas. RESULTS By maximally sampling the tumor geography without excluding samples based on low cancer cell fraction (CCF), we identify a subpopulation of glioblastoma with pervasively lower CCF likely excluded by other atlases, such as the TCGA, that used stringent CCF cutoffs. Exome sequencing of 3D-mapped samples from lower-grade tumors revealed that clonal expansions are typically spatially segregated, implying minimal tumor-wide intermixing of genetically heterogenous cells. Heterogeneity is less spatially segregated for faster-growing high-grade tumors, suggesting that cell populations expand in these tumors differently. Recurrent low-grade tumors have greater intratumoral mutational heterogeneity than newly diagnosed tumors, though this did not translate into greater dissimilarity in gene expression profiles for recurrent tumors, suggesting minimal functional impact of this additional mutational diversity on gene expression. CONCLUSIONS The delineation of spatial patterns of heterogeneity that our work provides enables more informed interpretation of biopsies and greater insight into the factors shaping intratumoral variation of gene expression patterns. Ongoing work is exploring the spatial patterning of amplification events and the tumor microenvironment.

Contains fulltext : 155226.pdf (Publisher’s version ) (Closed access) Temozolomide (TMZ) increases the overall survival of patients with glioblastoma (GBM), but its role in the clinical management of diffuse low-grade gliomas (LGG) is still being defined. DNA hypermethylation of the O (6) -methylguanine-DNA methyltransferase (MGMT) promoter is associated with an improved response to TMZ treatment, while inactivation of the DNA mismatch repair (MMR) pathway is associated with therapeutic resistance and TMZ-induced mutagenesis. We previously demonstrated that TMZ treatment of LGG induces driver mutations in the RB and AKT-mTOR pathways, which may drive malignant progression to secondary GBM. To better understand the mechanisms underlying TMZ-induced mutagenesis and malignant progression, we explored the evolution of MGMT methylation and genetic alterations affecting MMR genes in a cohort of 34 treatment-naive LGGs and their recurrences. Recurrences with TMZ-associated hypermutation had increased MGMT methylation compared to their untreated initial tumors and higher overall MGMT methylation compared to TMZ-treated non-hypermutated recurrences. A TMZ-associated mutation in one or more MMR genes was observed in five out of six TMZ-treated hypermutated recurrences. In two cases, pre-existing heterozygous deletions encompassing MGMT, or an MMR gene, were followed by TMZ-associated mutations in one of the genes of interest. These results suggest that tumor cells with methylated MGMT may undergo positive selection during TMZ treatment in the context of MMR deficiency.

SummaryThe evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.

Most cancers result from multiple clonal expansion events, resulting in tumors comprised of many subclones. The coexistence of these genetically distinct populations within a single tumor is thought to underlie the failure of most current treatments. Ascertaining how diverse populations of cells evolve within tumors, and the functional implications of this diversity, are the next steps to developing more informed treatments that overcome the barrier currently posed by intratumoral heterogeneity (ITH). Previous studies of adult diffuse glioma have been restricted in their ability to provide a detailed understanding of the spatial evolution of solid tumors due to insufficient sampling, typically just one biopsy per patient, and a lack of knowledge of where within the heterogeneous tumor the sample was obtained. We have developed a unique sample collection protocol, in which we record the precise, three-dimensional (3D) coordinate for each of 10 spatially-distinct samples obtained from each patient’s tumor. Whole exome data from IDH1 mutant grade II astrocytomas analyzed in this manner have revealed that expansion events are typically spatially segregated, suggesting minimal tumor-wide intermixing of subclonal cell populations in these low grade gliomas. Expression profiling of one particular case has furthermore revealed ITH of the immune microenvironment, uncovering a region of increased PD-L1 expression and IFN-gamma signaling. Ongoing analysis is focused on integrating genetic and expression profiles from these and additional spatially-mapped cases. This integrative analysis aims to dissect the relative contributions of subclonal variation and location within the tumor on gene expression patterns and ITH of the immune microenvironment. The delineation of the spatial patterns of ITH our work provides will enable more informed selection of biopsy targets in the future. It also lends greater insight into the factors shaping intratumoral variation of gene expression patterns and the immune microenvironment in adult diffuse glioma.

Background Rare multicentric lower-grade gliomas (LGGs) represent a unique opportunity to study the heterogeneity among distinct tumor foci in a single patient and to infer their origins and parallel patterns of evolution. Methods In this study, we integrate clinical features, histology, and immunohistochemistry for 4 patients with multicentric LGG, arising both synchronously and metachronously. For 3 patients we analyze the phylogeny of the lesions using exome sequencing, including one case with a total of 8 samples from the 2 lesions. Results One patient was diagnosed with multicentric isocitrate dehydrogenase 1 (IDH1) mutated diffuse astrocytomas harboring distinct IDH1 mutations, R132H and R132C; the latter mutation has been associated with Li-Fraumeni syndrome, which was subsequently confirmed in the patient's germline DNA and shown in additional cases with The Cancer Genome Atlas data. In another patient, phylogenetic analysis of synchronously arising grade II and grade III diffuse astrocytomas demonstrated a single shared mutation, IDH1 R132H, and revealed convergent evolution via non-overlapping mutations in ATRX and TP53. In 2 cases, there was divergent evolution of IDH1-mutated and 1p/19q-codeleted oligodendroglioma and IDH1-mutated and 1p/19q-intact diffuse astrocytoma, occurring synchronously in one case and metachronously in a second. Conclusions Each tumor in multicentric LGG cases may arise independently or may diverge very early in their development, presenting as genetically and histologically distinct tumors. Comprehensive sampling of these lesions can therefore significantly alter diagnosis and management. Additionally, somatic IDH1 R132C mutation in either multicentric or solitary LGG identifies unsuspected germline TP53 mutation, validating the limited number of published cases.

Low-grade gliomas (LGGs), which include grade II astrocytoma and grade II oligodendroglioma, inevitably recur despite aggressive treatment with surgery, and sometimes, with radiation and the chemotherapeutic agent temozolomide (TMZ). The clinical benefit of TMZ in LGG is unclear, and a subset of TMZ-treated LGGs recur with hypermutation in association with malignant progression to high-grade tumors. It is currently unknown why some TMZ-treated LGGs recur with hypermutation while others do not. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that reverses mutagenic lesions induced by TMZ. The amount of MGMT protein in a cell is regulated at the epigenetic level by promoter methylation. Here, we hypothesize that epigenetic silencing of MGMT by promoter methylation facilitates TMZ-induced mutagenesis and contributes to the development of hypermutation. We demonstrate in a cohort of 37 TMZ-treated patients with an initial diagnosis of IDH-mutant LGG that methylation level of the MGMT promoter in initial untreated tumors is significantly associated with hypermutation at recurrence. We also confirm our previous finding that methylation level of the MGMT promoter in recurrent hypermutated tumors is higher than in recurrent tumors that are not hypermutated. These results provide a plausible mechanistic basis for observed differences in propensity of TMZ-treated LGG patients to develop hypermutation at recurrence. Furthermore, they establish the potential of MGMT promoter methylation level to inform treatment decisions in the clinic for patients with newly diagnosed LGG.

Human organic cation transporter 3 (OCT3 and SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer, and is markedly underexpressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency 11.5% in African American) and g.-2G>A (rs555754) (minor allele frequency>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor-binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 messenger RNA expression levels were significantly higher in Asian (P

While patients with primary glioblastoma (GBM) have benefited from increased overall survival due to treatment with the alkylating chemotherapy temozolomide (TMZ), the benefit of TMZ for patients with diffuse low-grade gliomas (LGG) remains unknown. We previously demonstrated that among ten TMZ-treated patients with initial LGG (IDH1 mutant, 1p19q intact), treatment induced hypermutation in the recurrent tumors of six patients, all six of whom underwent malignant progression to secondary GBM (sGBM) (Johnson & Mazor, Science, 2014). To further explore the relationships among TMZ treatment, hypermutation and malignant progression, we studied tumor evolution in an expanded cohort of 35 TMZ-treated patients by exome-sequencing of paired IDH1/2-mutant LGGs and their post-TMZ recurrences. This expanded cohort allowed us to contrast patterns of hypermutation between 1p19q intact versus co-deleted subgroups. Hypermutated recurrences were common in both subgroups and consistently underwent malignant progression to the highest grade while acquiring somatic mutations in mismatch repair (MMR) genes. In contrast to the 1p19q intact subgroup in which TP53 mutations were present at diagnosis, the hypermutated recurrences of the 1p19q co-deleted subgroup frequently acquired TMZ-associated mutations in TP53. This suggests that TP53 mutations may cooperate with MMR mutations to convert TMZ-induced cytotoxicity to mutagenicity. Once hypermutated clones arose, we found that they dominated subsequent recurrences. In one case, a hypermutated sGBM was resected, but soon recurred with leptomeningeal spread, at which point hypermutated clones were found in the spinal cord. We examined the clonality of hypermutated tumors through exome-sequencing of multiple spatially distinct samples, from which we estimate that hypermutated recurrent tumors can originate from a single initiating cell. Together, these findings further our understanding of the molecular features and clinical behavior of hypermutated clones, raising concerns about the potent mutagenic activity of TMZ and other alkylating agents in LGG patients.

The human telomerase is an enzyme critical for maintaining telomere length and chromosomal stability in stem cells. The transcriptional regulation of the telomerase reverse transcriptase (TERT) gene, encoding the catalytic subunit of telomerase, is a rate-limiting step in modulating telomerase activity. Although normally silenced in somatic cells, TERT is aberrantly expressed in 90% of aggressive cancers, highlighting this event as a hallmark of tumorigenesis. Reactivating telomerase helps cells with finite lifespan to achieve limitless proliferative potential and bypass cellular senescence induced by DNA replication-associated telomere shortening. Recently discovered non-coding mutations in the TERT promoter are among the most common genetic alterations observed across multiple cancer types. Specifically, one of two positions, G228A or G250A, is mutated in 20% of medulloblastomas, 44% of hepatocellular carcinomas (HCC), 66% of urothelial carcinomas of the bladder, 71% of melanomas, 78% of oligodendrogliomas, and 83% of primary glioblastomas (GBM). The common mutation sites may create de-novo ETS family transcription factor binding sites, but the precise mechanism of how these mutations confer increased TERT expression has remained elusive. Here, a focused siRNA screen of the many ETS transcription factors expressed in GBM identifies GABPA as the single ETS factor to selectively regulate the mutant but not the wild type TERT promoter. Single molecule binding assays and ChIP-qPCR analysis reveal that GABPA is exclusively recruited to the mutant allele in vitro and in vivo respectively. Furthermore, this allelic recruitment is consistent across four tested cancer types, highlighting a shared mechanism underlying mutant TERT promoter activation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression across multiple cancers.

Studies have linked the consumption of broccoli and other cruciferous vegetables to a reduced risk of breast cancer. The phytochemical indole-3-carbinol (I3C), present in cruciferous vegetables, and its major acid-catalyzed reaction product 3,3'-diindolylmethane (DIM) have bioactivities relevant to the inhibition of carcinogenesis. In this study, the effect of DIM on angiogenesis and tumorigenesis in a rodent model was investigated. We found that DIM produced a concentration-dependent decrease in proliferation, migration, invasion and capillary tube formation of cultured human umbilical vein endothelial cells (HUVECs). Consistent with its antiproliferative effect, which was significant at only 5 microM DIM, this indole caused a G1 cell cycle arrest in actively proliferating HUVECs. Furthermore, DIM downregulated the expression of cyclin-dependent kinases 2 and 6 (CDK2, CDK6), and upregulated the expression of CDK inhibitor, p27(Kip1), in HUVECs. We observed further in a complementary in vivo Matrigel plug angiogenesis assay that, compared with vehicle control, neovascularization was inhibited up to 76% following the administration of 5 mg/kg DIM to female C57BL/6 mice. Finally, this dose of DIM also inhibited the growth of human MCF-7 cell tumor xenografts by up to 64% in female athymic (nu/nu) mice, compared with the vehicle control. This is the first study to show that DIM can strongly inhibit the development of human breast tumor in a xenograft model and to provide evidence for the antiangiogenic properties of this dietary indole.

The clonal evolution of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast, tumor epigenetic states, including DNA methylation, are reversible and sensitive to the tumor microenvironment, presumably precluding the use of epigenetics to discover tumor phylogeny. Here we examined the spatial and temporal dynamics of DNA methylation in a clinically and genetically characterized cohort of IDH1-mutant low-grade gliomas and their patient-matched recurrences. WHO grade II gliomas are diffuse, infiltrative tumors that frequently recur and may undergo malignant progression to a higher grade with a worse prognosis. The extent to which epigenetic alterations contribute to the evolution of low-grade gliomas, including malignant progression, is unknown. While all gliomas in the cohort exhibited the hypermethylation signature associated with IDH1 mutation, low-grade gliomas that underwent malignant progression to high-grade glioblastoma (GBM) had a unique signature of DNA hypomethylation enriched for active enhancers, as well as sites of age-related hypermethylation in the brain. Genes with promoter hypomethylation and concordant transcriptional upregulation during evolution to GBM were enriched in cell cycle function, evolving in concert with genetic alterations that deregulate the G1/S cell cycle checkpoint. Despite the plasticity of tumor epigenetic states, phyloepigenetic trees robustly recapitulated phylogenetic trees derived from somatic mutations in the same patients. These findings highlight widespread co-dependency of genetic and epigenetic events throughout the clonal evolution of initial and recurrent glioma.

Pancreatic ductal adenocarcinoma (PDAC) is usually incurable. Contrary to genetic mechanisms involved in PDAC pathogenesis, epigenetic alterations are ill defined. Here, we determine the contribution of epigenetically silenced genes to the development of PDAC. We analyzed enriched, highly methylated DNAs from PDACs, chronic pancreatitis (CP) and normal tissues using CpG island microarrays and identified WNK2 as a prominent candidate tumor suppressor gene being downregulated early in PDAC development. WNK2 was further investigated in tissue microarrays, methylation analysis of early pancreatic intraepithelial neoplasia (PanIN), mouse models for PDAC and pancreatitis, re-expression studies after demethylation, and cell growth assays using WNK2 overexpression. Demethylation assays confirmed the link between methylation and expression. WNK2 hypermethylation was higher in tumor than in surrounding inflamed tissues and was observed in PanIN lesions as well as in a PDAC mouse model. WNK2 mRNA and protein expressions were lower in PDAC and CP compared with normal tissues both in patients and mouse models. Overexpression of WNK2 led to reduced cell growth, and WNK2 expression in tissues correlated negatively with pERK1/2 expression, a downstream target of WNK2 responsible for cell proliferation. Downregulation of WNK2 by promoter hypermethylation occurs early in PDAC pathogenesis and may support tumor cell growth via the ERK–MAPK pathway.

Back with a Vengeance After surgery, gliomas (a type of brain tumor) recur in nearly all patients and often in a more aggressive form. Johnson et al. (p. 189 , published online 12 December 2013) used exome sequencing to explore whether recurrent tumors harbor different mutations than the primary tumors and whether the mutational profile in the recurrences is influenced by postsurgical treatment of patients with temozolomide (TMZ), a chemotherapeutic drug known to damage DNA. In more than 40% of cases, at least half of the mutations in the initial glioma were undetected at recurrence. The recurrent tumors in many of the TMZ-treated patients bore the signature of TMZ-induced mutagenesis and appeared to follow an evolutionary path to high-grade glioma distinct from that in untreated patients.

Human induced pluripotent stem (iPS) cells are remarkably similar to embryonic stem (ES) cells, but recent reports indicate that there may be important differences between them. We carried out a systematic comparison of human iPS cells generated from hepatocytes (representative of endoderm), skin fibroblasts (mesoderm) and melanocytes (ectoderm). All low-passage iPS cells analysed retain a transcriptional memory of the original cells. The persistent expression of somatic genes can be partially explained by incomplete promoter DNA methylation. This epigenetic mechanism underlies a robust form of memory that can be found in iPS cells generated by multiple laboratories using different methods, including RNA transfection. Incompletely silenced genes tend to be isolated from other genes that are repressed during reprogramming, indicating that recruitment of the silencing machinery may be inefficient at isolated genes. Knockdown of the incompletely reprogrammed gene C9orf64 (chromosome 9 open reading frame 64) reduces the efficiency of human iPS cell generation, indicating that somatic memory genes may be functionally relevant during reprogramming.

Purpose: To address the association between sequence variants within the MGMT (O6-methylguanine-DNA methyltransferase) promoter–enhancer region and methylation of MGMT in premalignant lesions from smokers and lung adenocarcinomas, their biological effects on gene regulation, and targeting MGMT for therapy. Experimental Design: Single nucleotide polymorphisms (SNP) identified through sequencing a 1.9 kb fragment 5′ of MGMT were examined in relation to MGMT methylation in 169 lung adenocarcinomas and 1,731 sputum samples from smokers. The effect of promoter haplotypes on MGMT expression was tested using a luciferase reporter assay and cDNA expression analysis along with allele-specific sequencing for methylation. The response of MGMT methylated lung cancer cell lines to the alkylating agent temozolomide (TMZ) was assessed. Results: The A allele of rs16906252 and the haplotype containing this SNP were strongly associated with increased risk for MGMT methylation in adenocarcinomas (ORs ≥ 94). This association was observed to a lesser extent in sputum samples in both smoker cohorts. The A allele was selectively methylated in primary lung tumors and cell lines heterozygous for rs16906252. With the most common haplotype as the reference, a 20 to 41% reduction in promoter activity was seen for the haplotype carrying the A allele that correlated with lower MGMT expression. The sensitivity of lung cancer cell lines to TMZ was strongly correlated with levels of MGMT methylation and expression. Conclusions: These studies provide strong evidence that the A allele of a MGMT promoter–enhancer SNP is a key determinant for MGMT methylation in lung carcinogenesis. Moreover, TMZ treatment may benefit a subset of lung cancer patients methylated for MGMT. Clin Cancer Res; 17(7); 2014–23. ©2011 AACR.

Extensive neuropil invasion is a hallmark of glioma growth and a subset of tumors demonstrate recurrence in the contralateral hemisphere without a path of tumor spread detected by MR imaging. We used an integrated genomic and radiographic approach to study a patient with a right nonenhancing insular mass histologically diagnosed as IDH1/2 wild-type anaplastic astrocytoma (AA) and a punctate nonenhancing T2 hyperintense focus within the left middle frontal gyrus subcortical white matter. Follow-up MR imaging, six months after initiation of Temozolomide based chemoradiotherapy, demonstrated focal enhancement within the left middle frontal gyrus lesion. Six months later a rim enhancing centrally necrotic mass involving left frontal lobe white matter was observed and diagnosed as glioblastoma. Whole exome sequencing of both tumors and targeted Sanger sequencing of 7-9 samplings from each tumor identified a novel five amino acid in-frame insertion in the PTEN C2 domain in both tumors that potentially disrupts cell membrane interaction and activates phosphotidylinositol 3-kinase (PI3K) signaling. As wild-type PTEN inhibits cellular migration, this early PTEN mutation might also promote invasion and the seeding of metastatic foci remote from the initial tumor mass. The right insular AA harbored amplifications of EGFR, MDM4 and PDGFRA (amplified and mutated) that were not detected within the contralateral glioblastoma. Conversely, the glioblastoma acquired CDKN2A homozygous deletion, probable NF1 biallelic inactivation (mutation and heterozygous loss), and a second hit to PTEN (heterozygous loss). Thirty-six shared mutations (including PTEN) and two shared copy number alterations unequivocally indicate a common origin for these spatially and temporally separated tumors. Taken together, these integrated radiographic and genomic data strongly suggest that the cells seeding the left frontal lobe tumor migrated from the evolving right insular tumor at an early time point and then followed an independent evolutionary trajectory.

Meningiomas are the second most common primary tumor of the CNS.1 They are thought to arise from neoplastic leptomeningeal cells lining the brain and spinal cord and are graded according to their histology using the WHO classification system.2 The majority of meningiomas are benign (grade I) tumors that are usually cured by surgical resection. Atypical (grade II) and malignant (grade III) meningiomas account for 10% of cases and carry a worse prognosis due to their higher likelihood of recurrence and metastasis.3 The mechanism of meningioma development remains enigmatic from a genetic standpoint. Although mutations of the gene for the hereditary disorder neurofibromatosis type 2 (NF2) are a defining alteration in meningiomas, only 60% of sporadic tumors carry this abnormality.4 Other genes involved in meningioma development have been sought, but identifying compelling candidates has proven challenging. One reason is that the complex chromosomal imbalances make positional cloning studies difficult.5,6 However, another reason may be that other mechanisms, such as epigenetic alterations, may affect gene expression. This possibility is suggested by the apparent absence of any large genetic alterations in nearly 40% of meningiomas.5,6 Loss of expression of components of the transforming growth factor-β signaling pathway distinguish high-proliferative from low-proliferative meningiomas,7 suggesting a link between specific alterations in gene expression and meningioma cell proliferation. Epigenetic mechanisms transcriptionally silence genes by methylating CpG islands within promoter regions,8 by altering the acetylation or methylation status of local histones, or both. Because these modifications preserve the normal genomic sequence, conventional genetic methods are unable to detect aberrant methylation or histone modifications. Gene-specific assays have identified aberrant methylation on CDKN2A, CDKN2B, DAPK, GSTP1, MGMT, p14, p17, RB1, THBS, NDRG2, and VHL in meningiomas.9–15 Although this single-gene approach demonstrates aberrant methylation in meningiomas, it is limited in that only a fraction of the 30,000 CpG islands are surveyed.16 To determine the extent of epigenetic involvement in meningiomas, larger-scale assessments of aberrant methylation are necessary. Restriction landmark genome scanning (RLGS) is a two-dimensional gel-based method for assessing the methylation status of thousands of CpG islands.17 When combined with array comparative genomic hybridization (aCGH),18 a high-resolution method of determining changes in copy number across the genome, the relative contributions of genetic and epigenetic mechanisms can be determined.19,20 In this study, we hypothesized that epigenetic mechanisms play a significant role in meningioma development and sought to determine their relative contribution in each malignancy grade.

Tumors arise in part from the deleterious effects of genetic and epigenetic mechanisms on gene expression. In several mouse models of human tumors, the tumorigenic phenotype is reversible, suggesting that epigenetic mechanisms also contribute significantly to tumorigenesis in mice. It is not known whether these are the same epigenetic mechanisms in human and mouse tumors or whether they affect homologous genes. Using an integrated approach for genome-wide methylation and copy number analyses, we identified SLC5A8 on chromosome 12q23.1 that was affected frequently by aberrant methylation in human astrocytomas and oligodendrogliomas. SLC5A8 encodes a sodium monocarboxylate cotransporter that was highly expressed in normal brain but was significant down-regulated in primary gliomas. Bisulfite sequencing analysis showed that the CpG island was unmethylated in normal brain but frequently localized methylated in brain tumors, consistent with the tumor-specific loss of gene expression. In glioma cell lines, SLC5A8 expression was also suppressed but could be reactivated with a methylation inhibitor. Expression of exogenous SLC5A8 in LN229 and LN443 glioma cells inhibited colony formation, suggesting that it may function as a growth suppressor in normal brain cells. Remarkably, 9 of 10 murine oligodendroglial tumors (from p53+/− or ink4a/arf+/− animals transgenic for S100β-v-erbB) showed a similar tumor-specific down-regulation of mSLC5A8, the highly conserved mouse homologue. Taken together, these data suggest that SLC5A8 functions as a growth suppressor gene in vitro and that it is silenced frequently by epigenetic mechanisms in primary gliomas. The shared epigenetic inactivation of mSLC5A8 in mouse gliomas indicates an additional degree of commonality in the origin and/or pathway to tumorigenesis between primary human tumors and these mouse models of gliomas.

3,3'-Diindolylmethane (DIM) is a major in vivo derivative of the putative anticancer agent indole-3-carbinol (I3C), which is present in vegetables of the Brassica genus. At concentrations above 10 microM, DIM inhibited DNA synthesis and cell proliferation in both estrogen receptor replete (MCF-7) and deficient (MDA-MB-231) human breast cancer cells in a concentration- and time-dependent manner. These antiproliferative effects were accompanied by characteristic indications of programmed cell death in both cell lines, including externalization of phosphatidylserine, chromatin condensation, and DNA fragmentation. Furthermore, Western and Northern blot analyses, as well as coimmunoprecipitation assays, revealed that in both MCF-7 and MDA-MB-231 cells, DIM treatment decreased total transcript and protein levels of the apoptosis inhibitory protein Bcl-2, and the amount of Bcl-2 bound to the pro-apoptotic protein Bax. DIM treatment also caused an increase in Bax protein levels, but did not affect the level of Bax that was bound to Bcl-2. As a functional test of the role of Bcl-2 down-regulation in the DIM-induced apoptotic response, ectopic expression of Bcl-2 in MCF-7 cells was shown to attenuate the apoptotic effect of DIM. These results demonstrate that DIM can induce apoptosis in breast cancer cells independent of estrogen receptor status by a process that is mediated by the modulated expression of the Bax/Bcl-2 family of apoptotic regulatory factors.

Low-grade gliomas frequently recur after surgical resection and may undergo malignant progression to a higher grade with a significantly worse prognosis. Understanding the origin and evolution of recurrences is critical for effectively treating residual disease to delay or prevent recurrence. Here, we extend previous work by sequencing the exomes of over 30 initial low-grade gliomas and their patient-matched recurrences to reconstruct the patterns of clonal evolution. We also sequence multiple spatially distinct samples from both initial low-grade and recurrent high-grade tumors to extensively dissect intratumoral heterogeneity. We identify a broad spectrum of genetic relatedness within tumor pairs, with the unexpected loss of canonical driver mutations upon recurrence. Therefore, resected portions of initial tumors can differ dramatically from tumorigenic portions of adjacent residual disease. The identification of common ancestral clones additionally reveals the order in which key driver mutations are acquired, allowing for a model of low-grade astrocytoma gliomagenesis. We have also previously shown that the treatment of low-grade gliomas with temozolomide induces hypermutation and alters the function of key cancer genes and their cognate pathways, potentially driving malignant progression. We extend that finding with an analysis of the evolution and intratumoral heterogeneity of additional hypermutated glioblastoma recurrences. These evolutionary trajectories have immediate ramifications for the use of mutagenic chemotherapies such as temozolomide, and for personalizing therapy to eliminate residual disease.