Single nucleus transcriptomics, pharmacokinetics, and pharmacodynamics of combined CDK4/6 and mTOR inhibition in a phase 0/1 trial of recurrent high-grade glioma.

Outcomes for adult patients with a high-grade glioma continue to be dismal and new treatment paradigms are urgently needed. To optimize the opportunity for discovery, we performed a phase 0/1 dose-escalation clinical trial that investigated tumor pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics following combined ribociclib (CDK4/6 inhibitor) and everolimus (mTOR inhibitor) treatment in recurrent high-grade glioma. Patients with a recurrent high-grade glioma (n = 24) harboring 1) CDKN2A / B deletion or CDK4 / 6 amplification, 2) PTEN loss or PIK3CA mutations, and 3) wild-type retinoblastoma protein (Rb) were enrolled. Patients received neoadjuvant ribociclib and everolimus treatment and no dose-limiting toxicities were observed. The median unbound ribociclib concentrations in Gadolinium non-enhancing tumor regions were 170 nM (range, 65 - 1770 nM) and 634 nM (range, 68 - 2345 nM) in patients receiving 5 days treatment at the daily dose of 400 and 600 mg, respectively. Unbound everolimus concentrations were below the limit of detection (< 0.1 nM) in both enhancing and non-enhancing tumor regions at all dose levels. We identified a significant decrease in MIB1 positive cells suggesting ribociclib-associated cell cycle inhibition. Single nuclei RNAseq (snRNA) based comparisons of 17 IDH-wild-type on-trial recurrences to 31 IDH-wild-type standard of care treated recurrences data demonstrated a significantly lower fraction of cycling and neural progenitor-like (NPC-like) malignant cell populations. We validated the CDK4/6 inhibitor-directed malignant cell state shifts using three patient-derived cell lines. The presented clinical trial highlights the value of integrating pharmacokinetics, pharmacodynamics, and single nucleus transcriptomics to assess treatment effects in phase 0/1 surgical tissues, including malignant cell state shifts. ClinicalTrials.gov identifier: NCT03834740 .

The Epigenetic Evolution of Glioma Is Determined by the IDH1 Mutation Status and Treatment Regimen.

Tumor adaptation or selection is thought to underlie therapy resistance in glioma. To investigate longitudinal epigenetic evolution of gliomas in response to therapeutic pressure, we performed an epigenomic analysis of 132 matched initial and recurrent tumors from patients with IDH-wildtype (IDHwt) and IDH-mutant (IDHmut) glioma. IDHwt gliomas showed a stable epigenome over time with relatively low levels of global methylation. The epigenome of IDHmut gliomas showed initial high levels of genome-wide DNA methylation that was progressively reduced to levels similar to those of IDHwt tumors. Integration of epigenomics, gene expression, and functional genomics identified HOXD13 as a master regulator of IDHmut astrocytoma evolution. Furthermore, relapse of IDHmut tumors was accompanied by histologic progression that was associated with survival, as validated in an independent cohort. Finally, the initial cell composition of the tumor microenvironment varied between IDHwt and IDHmut tumors and changed differentially following treatment, suggesting increased neoangiogenesis and T-cell infiltration upon treatment of IDHmut gliomas. This study provides one of the largest cohorts of paired longitudinal glioma samples with epigenomic, transcriptomic, and genomic profiling and suggests that treatment of IDHmut glioma is associated with epigenomic evolution toward an IDHwt-like phenotype. SIGNIFICANCE: Standard treatments are related to loss of DNA methylation in IDHmut glioma, resulting in epigenetic activation of genes associated with tumor progression and alterations in the microenvironment that resemble treatment-naïve IDHwt glioma.

Glioma progression is shaped by genetic evolution and microenvironment interactions.

The factors driving therapy resistance in diffuse glioma remain poorly understood. To identify treatment-associated cellular and genetic changes, we analyzed RNA and/or DNA sequencing data from the temporally separated tumor pairs of 304 adult patients with isocitrate dehydrogenase (IDH)-wild-type and IDH-mutant glioma. Tumors recurred in distinct manners that were dependent on IDH mutation status and attributable to changes in histological feature composition, somatic alterations, and microenvironment interactions. Hypermutation and acquired CDKN2A deletions were associated with an increase in proliferating neoplastic cells at recurrence in both glioma subtypes, reflecting active tumor growth. IDH-wild-type tumors were more invasive at recurrence, and their neoplastic cells exhibited increased expression of neuronal signaling programs that reflected a possible role for neuronal interactions in promoting glioma progression. Mesenchymal transition was associated with the presence of a myeloid cell state defined by specific ligand-receptor interactions with neoplastic cells. Collectively, these recurrence-associated phenotypes represent potential targets to alter disease progression.

Live-Cell Imaging Shows Uneven Segregation of Extrachromosomal DNA Elements and Transcriptionally Active Extrachromosomal DNA Hubs in Cancer.

Oncogenic extrachromosomal DNA elements (ecDNA) play an important role in tumor evolution, but our understanding of ecDNA biology is limited. We determined the distribution of single-cell ecDNA copy number across patient tissues and cell line models and observed how cell-to-cell ecDNA frequency varies greatly. The exceptional intratumoral heterogeneity of ecDNA suggested ecDNA-specific replication and propagation mechanisms. To evaluate the transfer of ecDNA genetic material from parental to offspring cells during mitosis, we established the CRISPR-based ecTag method. ecTag leverages ecDNA-specific breakpoint sequences to tag ecDNA with fluorescent markers in living cells. Applying ecTag during mitosis revealed disjointed ecDNA inheritance patterns, enabling rapid ecDNA accumulation in individual cells. After mitosis, ecDNAs clustered into ecDNA hubs, and ecDNA hubs colocalized with RNA polymerase II, promoting transcription of cargo oncogenes. Our observations provide direct evidence for uneven segregation of ecDNA and shed new light on mechanisms through which ecDNAs contribute to oncogenesis. SIGNIFICANCE: ecDNAs are vehicles for oncogene amplification. The circular nature of ecDNA affords unique properties, such as mobility and ecDNA-specific replication and segregation behavior. We uncovered fundamental ecDNA properties by tracking ecDNAs in live cells, highlighting uneven and random segregation and ecDNA hubs that drive cargo gene transcription.See related commentary by Henssen, p. 293.This article is highlighted in the In This Issue feature, p. 275.

Single-cell multimodal glioma analyses identify epigenetic regulators of cellular plasticity and environmental stress response.

Glioma intratumoral heterogeneity enables adaptation to challenging microenvironments and contributes to therapeutic resistance. We integrated 914 single-cell DNA methylomes, 55,284 single-cell transcriptomes and bulk multi-omic profiles across 11 adult IDH mutant or IDH wild-type gliomas to delineate sources of intratumoral heterogeneity. We showed that local DNA methylation disorder is associated with cell-cell DNA methylation differences, is elevated in more aggressive tumors, links with transcriptional disruption and is altered during the environmental stress response. Glioma cells under in vitro hypoxic and irradiation stress increased local DNA methylation disorder and shifted cell states. We identified a positive association between genetic and epigenetic instability that was supported in bulk longitudinally collected DNA methylation data. Increased DNA methylation disorder associated with accelerated disease progression and recurrently selected DNA methylation changes were enriched for environmental stress response pathways. Our work identified an epigenetically facilitated adaptive stress response process and highlights the importance of epigenetic heterogeneity in shaping therapeutic outcomes.

Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer.

Ionizing radiation causes DNA damage and is a mainstay for cancer treatment, but understanding of its genomic impact is limited. We analyzed mutational spectra following radiotherapy in 190 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium and 3,693 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified radiotherapy-associated significant increases in the burden of small deletions (5-15 bp) and large deletions (20+ bp to chromosome-arm length). Small deletions were characterized by a larger span size, lacking breakpoint microhomology and were genomically more dispersed when compared to pre-existing deletions and deletions in non-irradiated tumors. Mutational signature analysis implicated classical non-homologous end-joining-mediated DNA damage repair and APOBEC mutagenesis following radiotherapy. A high radiation-associated deletion burden was associated with worse clinical outcomes, suggesting that effective repair of radiation-induced DNA damage is detrimental to patient survival. These results may be leveraged to predict sensitivity to radiation therapy in recurrent cancer.

Spatial concordance of DNA methylation classification in diffuse glioma.

BACKGROUND: Intratumoral heterogeneity is a hallmark of diffuse gliomas. DNA methylation profiling is an emerging approach in the clinical classification of brain tumors. The goal of this study is to investigate the effects of intratumoral heterogeneity on classification confidence. METHODS: We used neuronavigation to acquire 133 image-guided and spatially separated stereotactic biopsy samples from 16 adult patients with a diffuse glioma (7 IDH-wildtype and 2 IDH-mutant glioblastoma, 6 diffuse astrocytoma, IDH-mutant and 1 oligodendroglioma, IDH-mutant and 1p19q codeleted), which we characterized using DNA methylation arrays. Samples were obtained from regions with and without abnormalities on contrast-enhanced T1-weighted and fluid-attenuated inversion recovery MRI. Methylation profiles were analyzed to devise a 3-dimensional reconstruction of (epi)genetic heterogeneity. Tumor purity was assessed from clonal methylation sites. RESULTS: Molecular aberrations indicated that tumor was found outside imaging abnormalities, underlining the infiltrative nature of this tumor and the limitations of current routine imaging modalities. We demonstrate that tumor purity is highly variable between samples and explains a substantial part of apparent epigenetic spatial heterogeneity. We observed that DNA methylation subtypes are often, but not always, conserved in space taking tumor purity and prediction accuracy into account. CONCLUSION: Our results underscore the infiltrative nature of diffuse gliomas and suggest that DNA methylation subtypes are relatively concordant in this tumor type, although some heterogeneity exists.

Comparative Molecular Life History of Spontaneous Canine and Human Gliomas.

Sporadic gliomas in companion dogs provide a window on the interaction between tumorigenic mechanisms and host environment. We compared the molecular profiles of canine gliomas with those of human pediatric and adult gliomas to characterize evolutionarily conserved mammalian mutational processes in gliomagenesis. Employing whole-genome, exome, transcriptome, and methylation sequencing of 83 canine gliomas, we found alterations shared between canine and human gliomas such as the receptor tyrosine kinases, TP53 and cell-cycle pathways, and IDH1 R132. Canine gliomas showed high similarity with human pediatric gliomas per robust aneuploidy, mutational rates, relative timing of mutations, and DNA-methylation patterns. Our cross-species comparative genomic analysis provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations.

Molecular and clonal evolution in recurrent metastatic gliosarcoma.

We discuss the molecular evolution of gliosarcoma, a mesenchymal type of glioblastoma (GBM), using the case of a 37-yr-old woman who developed two recurrences and an extracranial metastasis. She was initially diagnosed with isocitrate dehydrogenase (IDH) wild-type gliosarcoma in the frontal lobe and treated with surgery followed by concurrent radiotherapy with temozolomide. Five months later the tumor recurred in the left frontal lobe, outside the initially resected area, and was treated with further surgery and radiotherapy. Six months later the patient developed a second left frontal recurrence and was again treated with surgery and radiotherapy. Six weeks later, further recurrence was observed in the brain and bone, and biopsy confirmed metastases in the pelvic bones. To understand the clonal relationships between the four tumor instances and the origin of metastasis, we performed whole-genome sequencing of the intracranial tumors and the tumor located in the right iliac bone. We compared their mutational and copy-number profiles and inferred the clonal phylogeny. The tumors harbored shared alterations in GBM driver genes, including mutations in TP53, NF1, and RB1, and CDKN2A deletion. Whole-genome doubling was identified in the first recurrence and the extracranial metastasis. Comparisons of the metastatic to intracranial tumors highlighted a high similarity in molecular profile but contrasting evidence regarding the origin of the metastasis. Subclonal reconstruction suggested a parallel evolution of the recurrent tumors, and that the metastatic tumor was largely derived from the first recurrence. We conclude that metastasis in glioma can be a late event in tumorigenesis.

Mechanistic modeling of gastrointestinal motility with integrated dissolution for simulating drug absorption.

A new computational method - the multiple moving plug (MMP) model - is described to simulate the effect of gastrointestinal motility and dissolution on the pharmacokinetic profile of any given drug. The method is physiologically more consistent with the experimental evidence that fluid exists in discrete plugs in the gastrointestinal tract, and therefore is more realistic than modeling the gastrointestinal tract as a series of compartments with first-order transfer. The number of plugs used in simulations, their gastric emptying times and volumes, and their residence times in the small intestine can be matched with experimental data on motility. In sample simulations, drug absorption from a series of fluid plugs emptied from the stomach at evenly spaced time intervals showed lower Cmax and higher Tmax than an equivalent dose emptied immediately as a single plug. To the extent that new techniques can establish typical ranges for the volumes of fluid emptied from the stomach and their respective timing, the MMP model may be able to predict the effect of gastric emptying on the variability seen in pharmacokinetic profiles. This could lead to an expanded safe space for the regulatory acceptance of formulations based on dissolution data.

Genome-wide characterization of cytosine-specific 5-hydroxymethylation in normal breast tissue.

Despite recent evidence that 5-hydroxymethylcytosine (5hmC) possesses roles in gene regulation distinct from 5-methylcytosine (5mC), relatively little is known regarding the functions of 5hmC in mammalian tissues. To address this issue, we utilized an approach combining both paired bisulfite (BS) and oxidative bisulfite (oxBS) DNA treatment, to resolve genome-wide patterns of 5hmC and 5mC in normal breast tissue from disease-free women. Although less abundant than 5mC, 5hmC was differentially distributed, and consistently enriched among breast-specific enhancers and transcriptionally active chromatin. In contrast, regulatory regions associated with transcriptional inactivity, such as heterochromatin and repressed Polycomb regions, were relatively depleted of 5hmC. Gene regions containing abundant 5hmC were significantly associated with lactate oxidation, immune cell function, and prolactin signaling pathways. Furthermore, genes containing abundant 5hmC were enriched among those actively transcribed in normal breast tissue. Finally, in independent data sets, normal breast tissue 5hmC was significantly enriched among CpG loci demonstrated to have altered methylation in pre-invasive breast cancer and invasive breast tumors. Primarily, our findings identify genomic loci containing abundant 5hmC in breast tissues and provide a genome-wide map of nucleotide-level 5hmC in normal breast tissue. Additionally, these data suggest 5hmC may participate in gene regulatory programs that are dysregulated during breast-related carcinogenesis.

Longitudinal molecular trajectories of diffuse glioma in adults.

The evolutionary processes that drive universal therapeutic resistance in adult patients with diffuse glioma remain unclear1,2. Here we analysed temporally separated DNA-sequencing data and matched clinical annotation from 222 adult patients with glioma. By analysing mutations and copy numbers across the three major subtypes of diffuse glioma, we found that driver genes detected at the initial stage of disease were retained at recurrence, whereas there was little evidence of recurrence-specific gene alterations. Treatment with alkylating agents resulted in a hypermutator phenotype at different rates across the glioma subtypes, and hypermutation was not associated with differences in overall survival. Acquired aneuploidy was frequently detected in recurrent gliomas and was characterized by IDH mutation but without co-deletion of chromosome arms 1p/19q, and further converged with acquired alterations in the cell cycle and poor outcomes. The clonal architecture of each tumour remained similar over time, but the presence of subclonal selection was associated with decreased survival. Finally, there were no differences in the levels of immunoediting between initial and recurrent gliomas. Collectively, our results suggest that the strongest selective pressures occur during early glioma development and that current therapies shape this evolution in a largely stochastic manner.

Evolving Insights into the Molecular Neuropathology of Diffuse Gliomas in Adults.

Recent advances in molecular analysis and genome sequencing have prompted a paradigm shift in neuropathology. This article discusses the discovery and clinical relevance of molecular biomarkers in diffuse gliomas in adults and how these biomarkers led to revision of the World Health Organization classification of these tumors. We relate progress in clinical classification to an overview of studies using molecular profiling to study gene expression and DNA methylation to categorize diffuse gliomas in adults and issues dealing with intratumoral heterogeneity. These efforts will refine the taxonomy of diffuse gliomas, facilitate selection of appropriate treatment regimens, and ultimately improve patient's lives.

Deconvolution of DNA methylation identifies differentially methylated gene regions on 1p36 across breast cancer subtypes.

Breast cancer is a complex disease consisting of four distinct molecular subtypes. DNA methylation-based (DNAm) studies in tumors are complicated further by disease heterogeneity. In the present study, we compared DNAm in breast tumors with normal-adjacent breast samples from The Cancer Genome Atlas (TCGA). We constructed models stratified by tumor stage and PAM50 molecular subtype and performed cell-type reference-free deconvolution to control for cellular heterogeneity. We identified nineteen differentially methylated gene regions (DMGRs) in early stage tumors across eleven genes (AGRN, C1orf170, FAM41C, FLJ39609, HES4, ISG15, KLHL17, NOC2L, PLEKHN1, SAMD11, WASH5P). These regions were consistently differentially methylated in every subtype and all implicated genes are localized to the chromosomal cytoband 1p36.3. Seventeen of these DMGRs were independently validated in a similar analysis of an external data set. The identification and validation of shared DNAm alterations across tumor subtypes in early stage tumors advances our understanding of common biology underlying breast carcinogenesis and may contribute to biomarker development. We also discuss evidence of the specific importance and potential function of 1p36 in cancer.

Normal breast tissue DNA methylation differences at regulatory elements are associated with the cancer risk factor age.

BACKGROUND: The underlying biological mechanisms through which epidemiologically defined breast cancer risk factors contribute to disease risk remain poorly understood. Identification of the molecular changes associated with cancer risk factors in normal tissues may aid in determining the earliest events of carcinogenesis and informing cancer prevention strategies. METHODS: Here we investigated the impact cancer risk factors have on the normal breast epigenome by analyzing DNA methylation genome-wide (Infinium 450 K array) in cancer-free women from the Susan G. Komen Tissue Bank (n = 100). We tested the relation of established breast cancer risk factors, age, body mass index, parity, and family history of disease, with DNA methylation adjusting for potential variation in cell-type proportions. RESULTS: We identified 787 cytosine-guanine dinucleotide (CpG) sites that demonstrated significant associations (Q value <0.01) with subject age. Notably, DNA methylation was not strongly associated with the other evaluated breast cancer risk factors. Age-related DNA methylation changes are primarily increases in methylation enriched at breast epithelial cell enhancer regions (P = 7.1E-20), and binding sites of chromatin remodelers (MYC and CTCF). We validated the age-related associations in two independent populations, using normal breast tissue samples (n = 18) and samples of normal tissue adjacent to tumor tissue (n = 97). The genomic regions classified as age-related were more likely to be regions altered in both pre-invasive (n = 40, P = 3.0E-03) and invasive breast tumors (n = 731, P = 1.1E-13). CONCLUSIONS: DNA methylation changes with age occur at regulatory regions, and are further exacerbated in cancer, suggesting that age influences breast cancer risk in part through its contribution to epigenetic dysregulation in normal breast tissue.

Concordance of DNA methylation profiles between breast core biopsy and surgical excision specimens containing ductal carcinoma in situ (DCIS).

The utility and reliability of assessing molecular biomarkers for translational applications on pre-operative core biopsy specimens assume consistency of molecular profiles with larger surgical specimens. Whether DNA methylation in ductal carcinoma in situ (DCIS), measured in core biopsy and surgical specimens are similar, remains unclear. Here, we compared genome-scale DNA methylation measured in matched core biopsy and surgical specimens from DCIS, including specific DNA methylation biomarkers of subsequent invasive cancer. DNA was extracted from guided 2mm cores of formalin fixed paraffin embedded (FFPE) specimens, bisulfite-modified, and measured on the Illumina HumanMethylation450 BeadChip. DNA methylation profiles of core biopsies exhibited high concordance with matched surgical specimens. Within-subject variability in DNA methylation was significantly lower than between-subject variability (all P<2.20E-16). In 641 CpGs whose methylation was related with increased hazard of invasive breast cancer, lower within-subject than between-subject variability was observed in 92.3% of the study participants (P<0.05). Between patient-matched core biopsy and surgical specimens, <0.6% of CpGs measured had changes in median DNA methylation >15%, and a pathway analysis of these CpGs indicated enrichment for genes related with wound healing. Our results indicate that DNA methylation measured in core biopsies are representative of the matched surgical specimens and suggest that DCIS biomarkers measured in core biopsies can inform clinical decision-making.

Integrative epigenetic and genetic pan-cancer somatic alteration portraits.

Genetic and epigenetic alterations are required for carcinogenesis and the mutation burden across tumor types has been investigated. Here, we investigate epigenetic alterations with a novel measure of global DNA methylation dysregulation, the methylation dysregulation index (MDI), across 14 cancer types in The Cancer Genome Atlas (TCGA) database. DNA methylation data-obtained using Illumina HumanMethylation450 BeadChip-was accessed from TCGA. We calculated the MDI in 14 tumor types (n = 5,592 tumors), using adjacent normal tissues (n = 701) from each tumor site. Copy number alteration, and mutation burden were retrieved from cBioportal (n = 5,152). We tested the relation of subject MDI across tumors and with age, gender, tumor stage, estimated tumor purity, and copy number alterations for both overall MDI and genomic-context-specific MDI. We also investigated the top most dysregulated loci shared across tumor types. There was a broad range of extent in methylation dysregulation across tumor types (P < 2.2E-16). However, a consistent pattern of methylation dysregulation stratified by genomic context was observed across tumor types where the highest dysregulation occurred at non-CpG island regions. Considering other summary measures of somatic alteration, MDI was correlated with copy number alterations but not with mutation burden. Using the top dysregulated CpG sites in common across tumors, 4 classes of cancer types were observed, and the functional consequences of these alterations to gene expression were confirmed. This work identified the global DNA methylation dysregulation patterns across 14 cancer types showing a higher impact for the non-CpG island areas. The most dysregulated loci across cancer types identified common clusters across cancer types that may have implications for future treatment and prevention measures.

5-Hydroxymethylcytosine localizes to enhancer elements and is associated with survival in glioblastoma patients.

Glioblastomas exhibit widespread molecular alterations including a highly distorted epigenome. Here, we resolve genome-wide 5-methylcytosine and 5-hydroxymethylcytosine in glioblastoma through parallel processing of DNA with bisulfite and oxidative bisulfite treatments. We apply a statistical algorithm to estimate 5-methylcytosine, 5-hydroxymethylcytosine and unmethylated proportions from methylation array data. We show that 5-hydroxymethylcytosine is depleted in glioblastoma compared with prefrontal cortex tissue. In addition, the genomic localization of 5-hydroxymethylcytosine in glioblastoma is associated with features of dynamic cell-identity regulation such as tissue-specific transcription and super-enhancers. Annotation of 5-hydroxymethylcytosine genomic distribution reveal significant associations with RNA regulatory processes, immune function, stem cell maintenance and binding sites of transcription factors that drive cellular proliferation. In addition, model-based clustering results indicate that patients with low-5-hydroxymethylcytosine patterns have significantly poorer overall survival. Our results demonstrate that 5-hydroxymethylcytosine patterns are strongly related with transcription, localizes to disease-critical genes and are associated with patient prognosis.