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Crucial transitions in cancer-including tumor initiation, local expansion, metastasis, and therapeutic resistance-involve complex interactions between cells within the dynamic tumor ecosystem. Transformative single-cell genomics technologies and spatial multiplex in situ methods now provide an opportunity to interrogate this complexity at unprecedented resolution. The Human Tumor Atlas Network (HTAN), part of the National Cancer Institute (NCI) Cancer Moonshot Initiative, will establish a clinical, experimental, computational, and organizational framework to generate informative and accessible three-dimensional atlases of cancer transitions for a diverse set of tumor types. This effort complements both ongoing efforts to map healthy organs and previous large-scale cancer genomics approaches focused on bulk sequencing at a single point in time. Generating single-cell, multiparametric, longitudinal atlases and integrating them with clinical outcomes should help identify novel predictive biomarkers and features as well as therapeutically relevant cell types, cell states, and cellular interactions across transitions. The resulting tumor atlases should have a profound impact on our understanding of cancer biology and have the potential to improve cancer detection, prevention, and therapeutic discovery for better precision-medicine treatments of cancer patients and those at risk for cancer.

Mechanisms of therapeutic resistance and vulnerability evolve in metastatic cancers as tumor cells and extrinsic microenvironmental influences change during treatment. To support the development of methods for identifying these mechanisms in individual people, here we present an omic and multidimensional spatial (OMS) atlas generated from four serial biopsies of an individual with metastatic breast cancer during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata that includes treatment times and doses, anatomic imaging, and blood-based response measurements to clinical and exploratory analyses, which includes comprehensive DNA, RNA, and protein profiles; images of multiplexed immunostaining; and 2- and 3-dimensional scanning electron micrographs. These data report aspects of heterogeneity and evolution of the cancer genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples of how integrative analyses of these data reveal potential mechanisms of response and resistance and suggest novel therapeutic vulnerabilities.

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.

Aberrant DNA hypomethylation may play an important role in the growth rate of glioblastoma (GBM), but the functional impact on transcription remains poorly understood. We assayed the GBM methylome with MeDIP-seq and MRE-seq, adjusting for copy number differences, in a small set of non-glioma CpG island methylator phenotype (non-G-CIMP) primary tumors. Recurrent hypomethylated loci were enriched within a region of chromosome 5p15 that is specified as a cancer amplicon and also encompasses TERT, encoding telomerase reverse transcriptase, which plays a critical role in tumorigenesis. Overall, 76 gene body promoters were recurrently hypomethylated, including TERT and the oncogenes GLI3 and TP73. Recurring hypomethylation also affected previously unannotated alternative promoters, and luciferase reporter assays for three of four of these promoters confirmed strong promoter activity in GBM cells. Histone H3 lysine 4 trimethylation (H3K4me3) ChIP-seq on tissue from the GBMs uncovered peaks that coincide precisely with tumor-specific decrease of DNA methylation at 200 loci, 133 of which are in gene bodies. Detailed investigation of TP73 and TERT gene body hypomethylation demonstrated increased expression of corresponding alternate transcripts, which in TP73 encodes a truncated p73 protein with oncogenic function and in TERT encodes a putative reverse transcriptase-null protein. Our findings suggest that recurring gene body promoter hypomethylation events, along with histone H3K4 trimethylation, alter the transcriptional landscape of GBM through the activation of a limited number of normally silenced promoters within gene bodies, in at least one case leading to expression of an oncogenic protein.

Comprehensive characterization of an individual’s cancer using multi-omic analyses and an expanding list of targeted therapies is providing an opportunity to uncover therapeutic vulnerabilities and rationally target multiple driving alterations in the tumor. To leverage this opportunity, a multi-disciplinary team is required to unravel the complexity of tumor behavior and genomic variant information, integrate data from multi-omic assays, and exploit potential synergies from combination therapy while avoiding toxicity, all of which occurs within the context of the patient’s clinical history and comorbidities. We report the preliminary experience of OHSU Knight Cancer Institute’s SMMART (Serial Measurements of Molecular and Architectural Responses to Therapy) Treatments Program Multi-omics Tumor Board (MTB). The role of the SMMART MTB, to date, has been to develop and optimize procedures in a feasibility protocol for future application in the clinical setting where we will provide personalized combinatorial treatment suggestions. Here we report our preliminary experience over the last 15 months with multi-disciplinary tumor boards for metastatic breast cancer. A deep analysis of data, in many cases from serial biopsies, enabled identification of personalized, multi-targeted therapy. Clinical and laboratory data are explored individually and collectively by oncologists, oncology pharmacists, molecular pathologists, cancer biologists, and computational biologists. Data from CLIA-certified analytics include, but are not limited to, a 124 gene targeted panel, whole exome sequencing, MSI, gene-fusion, RNAseq, and clinical IHC assays that provide status information about receptors (ER/PR/AR/HER2), proliferation rate, and tumor immunogenicity along with reflexive assays for expression of relevant proteins such as p16, RB, and PTEN. A customized LabKey® system allows for visual display of a patient’s clinical timeline with information about diagnoses, treatments, biopsies events, radiographic changes, and blood biomarkers; thereby providing MTB participants with a holistic view of the patient’s case summary. Our experience with several example breast cancer case scenarios identified therapeutic vulnerabilities that would not have been considered by a standard clinical tumor board with genomic data alone. Recent cases have included observations such as HER2 status switching, a rare pathogenic germline mutation, high PD-L1 expression, and gained expression of the androgen receptor. These cases further emphasize the value and importance of MTB discussions to oncologists for interpreting and analyzing complex multi-omic data and uncovering therapeutic options for patients. This experience will be utilized in the future for employing SMMART MTBs in a clinical setting as a platform to triage patients into different multi-targeted combinatorial treatment options. Citation Format: Jamie M. Keck, Swapnil Parmar, Ben Kong, Zahi Mitri, Christopher Corless, Annette Kolodzie, Allison Creason, Jeremy Goecks, Patrick Leyshock, Kiara Siex, Brett E. Johnson, Janice Patterson, Laura Heiser, Anastasiya Olson, Matt Viehdorfer, Georgia Mayfield, Jennifer Laverdure, Joe W. Gray, Gordon B. Mills, Raymond C. Bergan. Leveraging multi-omics tumor boards for precision medicine in the OHSU SMMART Treatments Program [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4929.

Rapid advances in molecular diagnostics have led to a growing need for sophisticated and adaptive biospecimen collection methodologies that employ careful acquisition, preservation, and allocation of limited biopsy tissue and blood specimens. To facilitate the comprehensive characterization of serial biospecimens from patients participating in real-time adaptive treatment precision medicine clinical trials, we designed a multi-omic and multi-imaging analytical platform and sought to maximize the quality and suitability of tissue for each downstream analytic. We optimized this biospecimen management system over 38 research-protocol driven biopsies on 33 patients with late stage metastatic breast, prostate, and pancreatic cancer. Importantly, no biopsy-related adverse events occurred. All tissue samples and derivatives therefrom were barcoded and tracked using a custom database built on the LabVantage platform. Here, we report successful strategies for coordination between medical and surgical oncology, interventional radiology, pathology, and clinical and research laboratories. We developed a biopsy prioritization schema that considers patient safety, site selection, procedure type, and amount of tissue required. We evaluated specimens from a variety of biopsy procedures, including radiologically-guided needle cores, as well as laparoscopic, video-assisted thoracoscopic, and excisional biopsies. Radiologically-guided biopsies using 18 gauge needles yielded a median of 5 (1 cm) cores with 70% tumor cellularity, and was the best substrate to rapidly allocate known quantities of tissue into a variety of preservation formats. We also examined enhanced biospecimen preservation methods that improved performance of downstream analytics. For example, optimization of the formalin fixation process included transferring tissue from bedside to formalin within 2 minutes, maintaining low temperature throughout fixation, and standardizing the time spent in formalin. These efforts resulted in recovery of higher quality RNA from FFPE blocks, less tissue required for RNA sequencing, and opened the door to immunofluorescent staining of phosphoproteomic markers. Our protocol uses several preservation methods, including: FFPE for immunohistochemical and cyclic immunofluorescent staining as well as targeted panel, whole exome, and transcriptomic sequencing; OCT freezing for multiple single cell sequencing methods; flash freezing for proteomic profiling; disaggregation of live cells for 2D and 3D culturing; and a specialized fixative for electron microscopy. In conclusion, we demonstrate the feasibility of collecting high quality biospecimens for a variety of downstream analytical platforms with diverse sample requirements. These methods can be used to comprehensively characterize limited amounts of valuable tissue in molecularly driven clinical trials. Citation Format: Brett E. Johnson, Swapnil Parmar, Kiara Siex, Anastasiya Olson, Jennifer Laverdure, Jamie Keck, Annette Kolodzie, Alexander R. Guimaraes, Christopher Corless, Joe Gray, Gordon Mills, Raymond Bergan. Biospecimen management for the serial measurement of molecular and architectural responses to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 480.

Although it is known that the methylation of DNA in 5' promoters suppresses gene expression, the role of DNA methylation in gene bodies is unclear. In mammals, tissue- and cell type-specific methylation is present in a small percentage of 5' CpG island (CGI) promoters, whereas a far greater proportion occurs across gene bodies, coinciding with highly conserved sequences. Tissue-specific intragenic methylation might reduce, or, paradoxically, enhance transcription elongation efficiency. Capped analysis of gene expression (CAGE) experiments also indicate that transcription commonly initiates within and between genes. To investigate the role of intragenic methylation, we generated a map of DNA methylation from the human brain encompassing 24.7 million of the 28 million CpG sites. From the dense, high-resolution coverage of CpG islands, the majority of methylated CpG islands were shown to be in intragenic and intergenic regions, whereas less than 3% of CpG islands in 5' promoters were methylated. The CpG islands in all three locations overlapped with RNA markers of transcription initiation, and unmethylated CpG islands also overlapped significantly with trimethylation of H3K4, a histone modification enriched at promoters. The general and CpG-island-specific patterns of methylation are conserved in mouse tissues. An in-depth investigation of the human SHANK3 locus and its mouse homologue demonstrated that this tissue-specific DNA methylation regulates intragenic promoter activity in vitro and in vivo. These methylation-regulated, alternative transcripts are expressed in a tissue- and cell type-specific manner, and are expressed differentially within a single cell type from distinct brain regions. These results support a major role for intragenic methylation in regulating cell context-specific alternative promoters in gene bodies.

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.

Localized accessibility of critical DNA sequences to the regulatory machinery is a key requirement for regulation of human genes. Here we describe a high-resolution, genome-scale approach for quantifying chromatin accessibility by measuring DNase I sensitivity as a continuous function of genome position using tiling DNA microarrays (DNase-array). We demonstrate this approach across 1% ( approximately 30 Mb) of the human genome, wherein we localized 2,690 classical DNase I hypersensitive sites with high sensitivity and specificity, and also mapped larger-scale patterns of chromatin architecture. DNase I hypersensitive sites exhibit marked aggregation around transcriptional start sites (TSSs), though the majority mark nonpromoter functional elements. We also developed a computational approach for visualizing higher-order features of chromatin structure. This revealed that human chromatin organization is dominated by large (100-500 kb) 'superclusters' of DNase I hypersensitive sites, which encompass both gene-rich and gene-poor regions. DNase-array is a powerful and straightforward approach for systematic exposition of the cis-regulatory architecture of complex genomes.

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.

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.

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.

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

We report the generation and analysis of functional data from multiple, diverse experiments performed on a targeted 1% of the human genome as part of the pilot phase of the ENCODE Project. These data have been further integrated and augmented by a number of evolutionary and computational analyses. Together, our results advance the collective knowledge about human genome function in several major areas. First, our studies provide convincing evidence that the genome is pervasively transcribed, such that the majority of its bases can be found in primary transcripts, including non-protein-coding transcripts, and those that extensively overlap one another. Second, systematic examination of transcriptional regulation has yielded new understanding about transcription start sites, including their relationship to specific regulatory sequences and features of chromatin accessibility and histone modification. Third, a more sophisticated view about chromatin structure has emerged, including its interrelationship with DNA replication and transcriptional regulation. Finally, integration of these new sources of information, in particular with respect to mammalian evolution based on inter- and intra-species sequence comparisons, has yielded novel mechanistic and evolutionary insights about the functional landscape of the human genome. Together, these studies are defining a path forward to pursue a more-comprehensive characterisation of human genome function.

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.