Accumulation Of Somatic Mutations Research Articles

Cancer mutational burden is shaped by G4 DNA, replication stress and mitochondrial dysfunction

A hallmark of cancer is genomic instability, which can enable cancer cells to evade therapeutic strategies. Here we employed a computational approach to uncover mechanisms underlying cancer mutational burden by focusing upon relationships between 1) translocation breakpoints and the thousands of G4 DNA-forming sequences within retrotransposons impacting transcription and exemplifying probable non-B DNA structures and 2) transcriptome profiling and cancer mutations. We determined the location and number of G4 DNA-forming sequences in the Genome Reference Consortium Human Build 38 and found a total of 358,605 covering ∼13.4 million bases. By analyzing >97,000 unique translocation breakpoints from the Catalogue Of Somatic Mutations In Cancer (COSMIC), we found that breakpoints are overrepresented at G4 DNA-forming sequences within hominid-specific SVA retrotransposons, and generally occur in tumors with mutations in tumor suppressor genes, such as TP53. Furthermore, correlation analyses between mRNA levels and exome mutational loads from The Cancer Genome Atlas (TCGA) encompassing >450,000 gene-mutation regressions revealed strong positive and negative associations, which depended upon tissue of origin. The strongest positive correlations originated from genes not listed as cancer genes in COSMIC; yet, these show strong predictive power for survival in most tumor types by Kaplan-Meier estimation. Thus, correlation analyses of DNA structure and gene expression with mutation loads complement and extend more traditional approaches to elucidate processes shaping genomic instability in cancer. The combined results point to G4 DNA, activation of cell cycle/DNA repair pathways, and mitochondrial dysfunction as three major factors driving the accumulation of somatic mutations in cancer cells.

Somatic mutations, DNA methylation, and expression of DNA repair genes in Arabidopsis thaliana treated with 5-azacytidine

An inhibitor of DNA methylation 5-azacytidine (5A) is a chemical analog of the nucleoside cytidine. This study investigated the influence of 5A-induced DNA hypomethylation on the accumulation of somatic DNA mutations (nucleotide substitutions, indels) in the Actin2 3′ untranslated region, nuclear internal transcribed spacer ITS1-5.8rRNA-ITS2, and the ribulose-1,5-bisphosphate carboxylase/oxygenase gene of Arabidopsis thaliana and analyzed concurrent changes in the expression of DNA methyltransferase and DNA repair genes. The 5A treatment (20 mg per 100 g of soil) decreased DNA methylation, and the detected 5A-induced demethylation was associated with the up-regulation of the DNA methyltransferase genes: chromomethylase AtCMT3, methyltransferase AtMETI, and domains rearranged methyltransferases AtDRM1 and AtDRM2. Cultivation of plants in the presence of 5A led to a considerable increase in the number of single nucleotide substitutions in the analyzed DNA regions of 5A-treated A. thaliana. The 5A treatment significantly increased the transcriptions of 7 DNA repair genes (endonuclease AtARP, DNA demethylases AtDME and AtDML2, DNA glycosylase AtMBD4, DNA damage-binding protein AtDDB1, and photolyases AtUVR2 and AtUVR3) out of the 17 analyzed genes from the base excision repair, nucleotide excision repair, and photoreactivation pathways. However, 5A decreased the transcription of DNA 3′-phosphatase AtZDP, DNA repair protein AtRad23a, mismatch repair proteins AtMsh2 and AtMsh3. It is possible that the changes in the transcription of the DNA repair genes contributed to the detected increase in the number of single nucleotide substitutions that accumulated in the 5A-treated A. thaliana. Taken together, the data indicate that there is an interaction between the processes of DNA methylation and mutation accumulation.

Abstract B124: Personalized peptide vaccination based on patient-individual tumor-specific variants induces T-cell responses in pediatric patients

Abstract Background: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Accumulation of somatic mutations is one characteristic feature of malignanT-cells. These single-nucleotide variants (SNVs) can lead to altered amino acid sequences of the translated proteins, which in turn can be presented by malignant cells as antigenic peptides on HLA molecules. A peptide vaccination to induce neoantigen-specific T-cell responses, therefore, is a promising and versatile immunotherapeutic approach for the treatment of malignant diseases. Such approaches were predominantly applied in malignancies with high mutational load in adult patients until now. We developed a vaccine design platform based on sequencing data generated from a cohort of acute lymphoblastic leukemia (ALL) patients and tested individualized peptide vaccines in pediatric patients suffering from a variety of tumors with low mutational profiles. Methods: Nonsynonymous mutations were identified by whole-exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. HLA binding peptides harboring the altered amino acids were subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan for the patients’ individual HLA type. Individual peptides for treatment attempts were produced by chemical synthesis and vaccination cocktails were formulated. The vaccination schedule was 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvants. Some patients received simultaneous checkpoint blockade treatment with pembrolizumab or nivolumab. Response to the vaccination was monitored by detection of T-cells recognizing the vaccinated peptides occurring over time in peripheral blood of the patients. Monitoring was performed for each vaccination time point by pre-stimulation with the peptides and subsequent intracellular cytokine staining (ICS) of T-cells and FACS analysis. Results: Whole-exome sequencing was performed for 25 patients to identify ALL-specific SNVs using a comparative bioinformatics pipeline. We found an average of 39.2 mutations per patient on DNA level, with an average validation rate of 47% by RNA sequencing. Based on these data, an average of 35.1 HLA binders could be predicted per patient. We applied our platform for 6 patients with various malignancies based on compassionate need and designed individual peptide vaccines. In all cases validated mutations could be identified and epitope prediction was performed for HLA Class I and II binders. In 6/6 patients a de novo induced T-cell response against the vaccinated mutated HLA-binding peptides was detectable. Combination therapy with PD-1 blockade and peptide vaccination was well tolerated. T-cell responses were predominantly, but not exclusively, CD4+-restricted. Encouraged by these findings, we started a phase I/II clinical trial in patients with primary/relapsed ALL with the aim to prevent relapse and show safety and immunogenicity of the personalized peptide vaccine. Conclusions: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Whole-exome sequencing of pediatric ALL patients is feasible and yields a small amount of tumor-specific mutations per patient. However, these few mutations are sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T-cell responses in patients. The universal character of individualized peptide vaccination allows for application in virtually any malignant disease, as well as combination therapy approaches. The concept is now translated to clinical application in a phase I/II clinical trial in ALL, started in 2016 (NCT03559413). Citation Format: Armin Rabsteyn, Christina Kyzirakos, Christopher Schroeder, Marc Sturm, Christopher Mohr, Jakob Matthes, Magdalena Feldhahn, Nicolas Casadei, Martin Ebinger, Stefan Stevanovic, Peter Bauer, Oliver Kohlbacher, Cecile Gouttefangeas, Juergen Schaefer, Hans-Georg Rammensee, Rupert Handgretinger, Peter Lang. Personalized peptide vaccination based on patient-individual tumor-specific variants induces T-cell responses in pediatric patients [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B124.

Synopsis: Special Issue on “Disruption of signaling homeostasis induced crosstalk in the carcinogenesis paradigmEpistemology of the origin of cancer”

It is increasingly evident that carcinogenesis, in the vast majority of cancers, cannot be explained simply through an accumulation of somatic mutations, or epigenetics, the stem cell theory, or the Warburg effect. Here, decades of thinking based on incorrect assumptions has resulted in an incorrect hypothesis on the origin of cancer. Many papers studying DNA, genetics, RNA, miRNA, proteomics, and epigenetics have increased our understanding of biology. Our paradigm, though more complex, is more reliable and plausible. It states that cancer originates from a disruption of homeostasis. This essential biological phenomenon, homeostasis, maintains the interrelationships of various signaling pathways and induced crosstalk which modify cellular functions together with the interactions of surrounding cells and structures such that the equilibrium lies towards the optimal health of the organism. This Special Issue “Disruption of signaling homeostasis induced crosstalk in the carcinogenesis paradigm Epistemology of the origin of cancer” provides compelling evidence that carcinogenesis is explained by a six-step sequence of events for the vast majority of cancers. These six steps include, (1) a pathogenic stimulus followed by (2) chronic inflammation, from which develops (3) fibrosis with associated remodeling in the cellular microenvironment. From these changes a (4) pre-cancerous niche develops which triggers the deployment of (5) a chronic stress escape strategy, and when this fails to resolve, and (6) the transition of a normal cell to a cancer cell occurs. This paradigm provides opportunities to move away from a symptom-oriented understanding of cancer and is much closer to a cause-based understanding, which opens the door for early preventative strategies to mitigate cancer as a disease, and to interdict metastases. This is underpinned by the fact that an independent recently published proof of this paradigm showed how a stimulus trigger the proposed multi-sequence cascade of events as abrupt involution-induced chronic inflammation, followed by fibrosis with remodeling, which describes the pre-cancerous niche followed by hyperplasia, metaplasia, and cancer.

Human Papillomavirus Carcinogenesis Mediated by APOBEC Mutagenesis

Persistent infection with oncogenic human papillomaviruses (HPVs) is necessary for the development of cervical cancer, although the accumulation of somatic mutations in the host genome is also required for the generation of invasive cervical cancer. Recent studies have demonstrated concomitant genetic changes in the HPV genome; however, their relevance to cervical carcinogenesis is poorly understood. Here we review our recent study investigating the within-host genetic diversity of HPV and its relationship with cervical cancer progression through deep sequencing analyses of viral whole-genome sequences in clinical specimens. Intriguingly, HPV genomes within individual clinical samples show an astonishingly high level of nucleotide variation across all histological grades of cervical lesions. Among the various substitution patterns, C-to-T and C-to-A substitutions are the predominant changes observed in the HPV genomes. Analysis of the trinucleotide context for substituted bases reveals that TpCpN (N is any nucleotide), which is a preferred target sequence for the cellular apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like (APOBEC) proteins, is the major target for C-to-T substitutions in the HPV genomes. These mutational signature analyses strongly imply that within-host HPV variations are mostly generated through APOBEC-mediated mutagenesis. Because the HPV oncogenes E6 and E7 harbor APOBEC-related mutations, we propose a potential role for APOBEC-mediated mutagenesis in cervical carcinogenesis.

Long Interspersed Nuclear Element 1 Retrotransposons Become Deregulated during the Development of Ovarian Cancer Precursor Lesions

There is growing evidence that most high-grade serous ovarian carcinomas likely arise from local dissemination of precursor lesions of the fallopian tube. Evolution of these lesions from early p53 signatures to latter-stage, serous tubal intraepithelial carcinomas (STICs) is characterized by cytologic atypia, accumulation of somatic mutations, and genomic instability, the etiologies of which remain unclear. Long interspersed element 1 (LINE-1) retrotransposon is expressed in many carcinomas, including high-grade serous ovarian carcinoma, where it contributes to genomic instability; however, the timing of LINE-1 activation during this evolution has yet to be elucidated. In this study, we assessed LINE-1 open reading frame 1 protein expression in 12 p53 signature lesions, 32 STICs, and 112 various types of ovarian cancers via immunohistochemical staining and examined LINE-1 promoter methylation in representative cases. We found that 78% and 57% of STICs, with and without concurrent ovarian carcinomas, respectively, exhibited intense LINE-1 immunoreactivity compared with adjacent, normal-appearing fallopian tube epithelium. Hypomethylation of the LINE-1 promoter was found in all STICs exhibiting overexpression. None of the 12 p53 signatures demonstrated significant LINE-1 expression. In ovarian cancer, 84 (75%) of 112 ovarian carcinomas overexpressed LINE-1. Our results indicate that LINE-1 retrotransposons often become deregulated during progression of ovarian cancer precursor lesions from the p53 signature to STIC stages and remain highly expressed in carcinoma.

Mechanisms Underlying Augmented Lymphomagenesis by LUBAC; Possible Accumulation of AID-Mediated Somatic Mutations

Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is characterized by constitutive activation of NF-κB signaling, where protein ubiquitination is involved in various steps. The linear ubiquitin chains specifically generated by the linear ubiquitin assembly complex (LUBAC) has recently drawn attention in NF-κB signaling. LUBAC is known to be involved in NF-κB activation and cell death protection by conjugating linear chains onto several substrates including NEMO. We have reported increased frequency of rare germline single-nucleotide polymorphisms (SNPs) of HOIP, the catalytic subunit of LUBAC in ABC-DLBCL patients (Yang et al., Cancer Discov, 2014). Although the SNPs enhanced ligase activity of LUBAC, precise mechanisms underlying augmented lymphomagenesis by LUBAC remains unknown.In order to consolidate the roles of LUBAC in the pathogenesis of ABC-DLBCL, a mouse strain, in which wild-type murine HOIP is constitutively expressed in B cells from as early as the pre-B cell stage (CD19-cre-HOIP) is generated. In CD19-cre-HOIP, the increase of HOIP elevated the amount of trimeric LUBAC composed of HOIP, HOIL-1L and SHARPIN, augmenting NF-κB activation mildly in B cells. TLR-induced proliferation is more prominent in CD19-cre-HOIP B cells than littermate control, indicating that MYD88-dependent signaling is facilitated by LUBAC. Although some of the aged CD19-cre-HOIP mice showed splenomegaly, no CD19-cre-HOIP mice developed lymphomas as far as examined. These results indicated that enhanced expression of HOIP leads to augmentation of TLR-mediated signaling and proliferation in B cells, but it cannot induce lymphomagenesis by itself.Importantly, majority of ABC-DLBCL found in patients with the HOIP SNPs had oncogenic MYD88 L265P mutation. To assess synergistic effects of LUBAC and MYD88 L265P, we generated two mice strains, expressing MYD88 L252P, an equivalent mutation of human L265P in mice alone (CD19-cre-MYD88LP) or MYD88 L252P and HOIP (CD19-cre-HOIP/MYD88LP) in a B-cell specific manner.In CD19-cre-MYD88LP B cells, enhanced phosphorylation of IRAK4, degradation of IκBα, and expression of NF-κB target genes were observed as a result of constitutive activation of MYD88 signaling. Constitutive MYD88 signaling in B cells also led to the expansion of B cell population and splenomegaly since early age. As reported previously (Knittel et al., Blood, 2016), B-cell specific expression of MYD88 L252P resulted in a reduced survival and introduction of a HOIP transgenic allele significantly shortened survival of mice expressing MYD88 L252P. Since clonal B-cell lymphomas displaying characteristics of DLBCL could be found in both CD19-cre-MYD88LP and CD19-cre-HOIP/MYD88LP mice, the augmented LUBAC activity promotes active MYD88-mediated lymphomagenesis of B cells.To clarify the mechanisms underlying LUBAC-mediated acceleration of B lymphomagenesis, whole-exome-sequencing analyses of lymphomas developed in these mice were performed. Many of the genes mutated in these lymphomas recapitulated those reported in human DLBCL, and alignment analyses suggested that majority of these mutations were identified as targets of aberrant somatic hypermutations induced by AID, which were especially prominent in the tumors developed in CD19-cre-HOIP/MYD88LP mice. These findings strongly implied that LUBAC enhances AID-mediated somatic mutations.Induced expression of AID was unlikely the major reason for increased mutation loads by LUBAC, since AID expression was elevated in B cells of both CD19-cre-HOIP/MYD88LP, and CD19-cre-MYD88LP mice almost equally. LUBAC has been reported involved in the protection of DNA damage-induced apoptosis. AID is known to induce double strand DNA break, and we hypothesized that LUBAC may protect cells from DNA damage-induced cell death caused by AID. To probe the effect of enhanced expression of LUBAC on protection from genotoxin-mediated apoptosis, we treated Jurkat cells with cisplatin and found that enforced expression of HOIP protected cells from cisplatin-induced cell death, implying that cells with augmented LUBAC activity have higher tolerance to genotoxic stress.In summary, the present results indicated that protection of B-cells from cell death underlies accelerating lymphomagenesis caused by augmented LUBAC activity via partial overcoming AID-mediated DNA damage, which leads to accumulation of somatic mutations. DisclosuresNishikori:Ono Pharmaceuticals: Research Funding; Eisai: Research Funding. Takaori-Kondo:Bristol-Myers Squibb: Honoraria; Celgene: Honoraria, Research Funding; Novartis: Honoraria; Janssen Pharmaceuticals: Honoraria; Pfizer: Honoraria.

Population sequencing reveals clonal diversity and ancestral inbreeding in the grapevine cultivar Chardonnay.

Chardonnay is the basis of some of the world’s most iconic wines and its success is underpinned by a historic program of clonal selection. There are numerous clones of Chardonnay available that exhibit differences in key viticultural and oenological traits that have arisen from the accumulation of somatic mutations during centuries of asexual propagation. However, the genetic variation that underlies these differences remains largely unknown. To address this knowledge gap, a high-quality, diploid-phased Chardonnay genome assembly was produced from single-molecule real time sequencing, and combined with re-sequencing data from 15 different Chardonnay clones. There were 1620 markers identified that distinguish the 15 clones. These markers were reliably used for clonal identification of independently sourced genomic material, as well as in identifying a potential genetic basis for some clonal phenotypic differences. The predicted parentage of the Chardonnay haplomes was elucidated by mapping sequence data from the predicted parents of Chardonnay (Gouais blanc and Pinot noir) against the Chardonnay reference genome. This enabled the detection of instances of heterosis, with differentially-expanded gene families being inherited from the parents of Chardonnay. Most surprisingly however, the patterns of nucleotide variation present in the Chardonnay genome indicate that Pinot noir and Gouais blanc share an extremely high degree of kinship that has resulted in the Chardonnay genome displaying characteristics that are indicative of inbreeding.

Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase γ.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited syndromes and the accumulation of somatic mtDNA mutations is implicated in aging and common diseases. However, the mechanisms that influence the frequency and pathogenicity of mtDNA mutations are poorly understood. To address this matter, we created a Drosophila mtDNA mutator strain expressing a proofreading-deficient form of the mitochondrial DNA polymerase. Mutator flies have a dramatically increased somatic mtDNA mutation frequency that correlates with the dosage of the proofreading-deficient polymerase. Mutator flies also exhibit mitochondrial dysfunction, shortened lifespan, a progressive locomotor deficit, and loss of dopaminergic neurons. Surprisingly, the frequency of nonsynonymous, pathogenic, and conserved-site mutations in mutator flies exceeded predictions of a neutral mutational model, indicating the existence of a positive selection mechanism that favors deleterious mtDNA variants. We propose from these findings that deleterious mtDNA mutations are overrepresented because they selectively evade quality control surveillance or because they are amplified through compensatory mitochondrial biogenesis.

Clonal hematopoiesis: Genes and underlying mechanisms in cardiovascular disease development.

The clonal hematopoiesis when occurring without hematologic abnormalities is defined as clonal hematopoiesis of indeterminate potential (CHIP). Aging causes accumulation of somatic mutations, and hematopoietic stem cells (HSCs) can develop clonal expansion of different lineages by these mutations. CHIP has a correlation with cancer and cardiovascular disease (CVD) through acquired mutations in genes. DNMT3A, TET2, ASXL1, and JAK2 genes as well as other genes are the most common somatic mutations causing CHIP and CVD in an older age. Other factors such as cholesterol level, laboratory tests and indexes also affect CVD. In addition, mutations in adenosine triphosphate-binding cassette transporters and also chronic stress in nervous system can result in HSCs proliferation and CVD. However, laboratory tests and indexes are not sensitive for CVD diagnosis. But the therapeutic interventions can be helpful to prevent CVD cases by targeting somatic mutations, chemokine receptors, and growth factors in HSCs. Also, new drugs can control CVD by targeting of cells and their signaling pathways in HSCs. Therefore, more investigations are needed and more questions should be answered for the relationship between CHIP and CVD as a challenging issue in future.

Recurrent mutations at estrogen receptor binding sites alter chromatin topology and distal gene expression in breast cancer

BackgroundThe mutational processes underlying non-coding cancer mutations and their biological significance in tumor evolution are poorly understood. To get better insights into the biological mechanisms of mutational processes in breast cancer, we integrate whole-genome level somatic mutations from breast cancer patients with chromatin states and transcription factor binding events.ResultsWe discover that a large fraction of non-coding somatic mutations in estrogen receptor (ER)-positive breast cancers are confined to ER binding sites. Notably, the highly mutated estrogen receptor binding sites are associated with more frequent chromatin loop contacts and the associated distal genes are expressed at higher level. To elucidate the functional significance of these non-coding mutations, we focus on two of the recurrently mutated estrogen receptor binding sites. Our bioinformatics and biochemical analysis suggest loss of DNA-protein interactions due to the recurrent mutations. Through CRISPR interference, we find that the recurrently mutated regulatory element at the LRRC3C-GSDMA locus impacts the expression of multiple distal genes. Using a CRISPR base editor, we show that the recurrent C→T conversion at the ZNF143 locus results in decreased TF binding, increased chromatin loop formation, and increased expression of multiple distal genes. This single point mutation mediates reduced response to estradiol-induced cell proliferation but increased resistance to tamoxifen-induced growth inhibition.ConclusionsOur data suggest that ER binding is associated with localized accumulation of somatic mutations, some of which affect chromatin architecture, distal gene expression, and cellular phenotypes in ER-positive breast cancer.

Resprouters, assisted by somatic mutations, are as genetically diverse as nonsprouters in the world's fire-prone ecosystems

In fire-prone environments worldwide, resprouters mostly regenerate vegetatively after fire, whereas non (re)sprouters are killed by fire and rely entirely on stored seeds (soil or canopy storage) for regeneration. This dichotomy in post-fire regeneration strategies is a key mechanism for controlling the reproductive characteristics, demography, and population genetic structure of plant species in fire-prone ecosystems. Nonsprouters are considered to have higher within-population genetic variation than resprouters due to greater opportunities for recombination via their much greater seed production and frequent generation turnover. Empirical studies that explore this hypothesis are rare and the results are mixed. We collated published studies reporting genetic diversity measures of plant species in fire-prone ecosystems of four Mediterranean-climate regions. Ninety-two species were identified with unambiguous information on postfire regeneration type and with adequate sample sizes. We found no significant differences in population genetic diversity and structure between resprouters and nonresprouters across diversity parameters and genetic marker types, taxon groups or geographic regions. We conclude that resprouters are at least as genetically diverse as nonsprouters. We tested the hypothesis that accumulation of somatic mutations is a possible mechanism for maintaining genetic diversity among resprouters, by screening for microsatellite DNA mutations in the resprouter, Banksia attenuata. Genotyping of leaf material collected from disparate parts of individual plants demonstrated the existence of 2–5 somatic mutations among eleven microsatellite loci. The buildup of somatic mutations in meristematic buds during a resprouter's long lifespan and the considerable potential for interpopulation gene dispersal among resprouters may be key factors that enable their genetic diversity to keep pace with that of nonsprouters. Thus, resprouters and nonsprouters are equally capable of responding to natural selection, and therefore possess a similar potential to adapt to changing environmental conditions.

Human glioblastoma arises from subventricular zone cells with low-level driver mutations.

Glioblastoma (GBM) is a devastating and incurable brain tumour, with a median overall survival of fifteenmonths1,2. Identifying the cell of origin that harbours mutations that drive GBM could provide a fundamental basis for understanding disease progression and developing new treatments. Given that the accumulation of somatic mutations has been implicated in gliomagenesis, studies have suggested that neural stem cells (NSCs), with their self-renewal and proliferative capacities, in the subventricular zone (SVZ) of the adult human brain may be the cells from which GBM originates3-5. However, there is a lack of direct genetic evidence from human patients with GBM4,6-10. Here we describe direct molecular genetic evidence from patient brain tissue and genome-edited mouse models that show astrocyte-like NSCs in the SVZ to be the cell of origin that contains the driver mutations of human GBM. First, we performed deep sequencing of triple-matched tissues, consisting of (i) normal SVZ tissue away from the tumour mass, (ii) tumour tissue, and (iii) normal cortical tissue (or blood), from 28 patients with isocitrate dehydrogenase (IDH) wild-type GBM or other types of brain tumour. We found that normal SVZ tissue away from the tumour in 56.3% of patients with wild-type IDH GBM contained low-level GBM driver mutations (down to approximately 1% of the mutational burden) that were observed at high levels in their matching tumours. Moreover, by single-cell sequencing and laser microdissection analysis of patient brain tissue and genome editing of a mouse model, we found that astrocyte-like NSCs that carry driver mutations migrate from the SVZ and lead to the development of high-grade malignant gliomas in distant brain regions. Together, our results show that NSCs in human SVZ tissue are the cells of origin that contain the driver mutations of GBM.

Levels and prognostic impact of circulating markers of inflammation, endothelial activation and extracellular matrix remodelling in patients with lung cancer and chronic obstructive pulmonary disease

BackgroundThe development of both chronic obstructive pulmonary disease (COPD) and lung cancer (LC) is influenced by smoking related chronic pulmonary inflammation caused by an excessive innate immune response to smoke exposure. In addition, the smoking induced formation of covalent bonds between the carcinogens and DNA and the accumulation of permanent somatic mutations in critical genes are important in the carcinogenic processes, and can also induce inflammatory responses.How chronic inflammation is mirrored by serum markers in COPD and LC and if these markers reflect prognosis in patients with LC is, however, largely unknown.MethodsSerum levels of 18 markers reflecting inflammation, endothelial activation and extracellular matrix remodelling were analysed in 207 patients with non-small lung carcinoma (NSCLC) before surgery and 42 COPD patients. 56% of the LC patients also suffered from COPD. The serum samples were analysed by enzyme immunoassays.ResultsSerum levels of OPG, PTX3, AXL, ALCAM, sCD163, CD147, CatS and DLL1 were significantly higher in patients with COPD as compared to patients with LC. High sTNFR1 levels were associated with improved progression free survival (PFS) and overall survival (OS) in LC patients with (PFS hazard ratio (HR) 0.49, OS HR 0.33) and without COPD (OS HR 0.30). High levels of OPG were associated with improved PFS (HR 0.17) and OS (HR 0.14) for LC with COPD. CRP was significantly associated with overall survival regardless of COPD status.ConclusionSeveral markers reflecting inflammation, endothelial activation and extracellular matrix remodelling are elevated in serum from patients with COPD compared to LC patients. Presence of COPD might influence the levels of circulating biomarkers. Some of these markers are also associated with prognosis.

Prediction of acute myeloid leukaemia risk in healthy individuals.

The incidence of acute myeloid leukaemia (AML) increases with age and mortality exceeds 90% when diagnosed after age 65. Most cases arise without a detectable prodrome and present with the acute complications of bone marrow failure1. The onset of such de novo AML cases is typically preceded by the accumulation of somatic mutations in pre-leukaemic haematopoietic stem and progenitor cells (HSPC) that undergo clonal expansion2,3. However, recurrent AML mutations also accumulate in HSPCs during ageing of healthy individuals who do not develop AML, a phenomenon referred to as age-related clonal haematopoiesis (ARCH),4–8. To distinguish individuals at high risk of developing AML from those with benign ARCH, we undertook deep sequencing of genes recurrently mutated in AML in the peripheral blood cells of 95 individuals sampled on average 6.3 years before AML diagnosis (pre-AML group), together with 414 unselected age- and gender-matched individuals (control group). Pre-AML cases were distinct from controls with more mutations per sample, higher variant allele frequencies (VAF) reflective of greater clonal expansion, and enrichment for mutations in specific genes. Genetic parameters were used to derive a model that accurately predicted AML-free survival; this model was validated in an independent cohort of 29 pre-AMLs and 262 controls. Since AML is rare, we also developed an AML predictive model using a large electronic health record database that identified individuals at greater risk. Collectively our findings provide a proof-of-concept that it is possible to discriminate ARCH from pre-AML many years prior to malignant transformation. This could in the future enable earlier detection, monitoring and potentially inform intervention.

Abstract B43: Germline and somatic mutations, immune signatures, and epigenetic alterations linked to neighborhood determinants of health differ in Black and non-Latino White head and neck cancer patients

Abstract Somatic mutations play an important role in cancer prognosis and can pave the way for precision medicine to overcome cancer disparities. Current models hold that solid tumors evolve from microscopic clonal cellular proliferations driven through progressive histologic stages by the acquisition of somatic alterations. The accumulation of somatic mutations is associated to varying rates of stem cell divisions, leading to replicative errors that arise in response to hereditary burden, environmental stressors, or random error. Environmental, lifestyle, and neighborhood factors track closely with the acquisition of DNA methylation alterations and may partly explain the differential accumulation of genetic alterations in cancer patients. However, the combined roles of germline and somatic mutation burden, promoter methylation, and neighborhood characteristics in cancer disparities are unknown, in large part because most of the genomic studies have been performed on genomes of European descendants. Here we show that neighborhood characteristics linked to genetic, epigenetic, and tumor-associated immune signatures differ in Black and non-Latino White (NLW) head and neck squamous cell carcinoma (HNSCC) patients. Promoter methylation in PAX genes is associated to neighborhood characteristics at the zip code level (p<0.05). We also show that the number of tumor-infiltrating lymphocytes (TILs), the frequency of TP53 mutations, and a C -> A germline mutation in JAK3, chr19:17954215 (protein P132T), differ in Black when compared to NLW HNSCC patients (p<0.05). Our results demonstrate that genetic, epigenetic, and immuno-oncology alterations differ in racially diverse HNSCC patients. TILs and JAK3 germline mutations also vary across sex and anatomic tumor site. Finally, we show that molecular alterations are linked to known neighborhood stressors at the zip code level. Together, these data suggest that paired molecular and social determinants of health at the neighborhood level can inform the delivery of precision oncology services to racial and ethnically diverse populations. Citation Format: Rafael Guerrero-Preston, Fahcina Lawson, Laura Manuel, Blanca Valle, Tal Hadar, Bianca Rivera, Oluwasina Folawiyo, Adriana Baez Bermejo, Luigi Marchionni, Wayne Koch, William Westra, David Sidransky. Germline and somatic mutations, immune signatures, and epigenetic alterations linked to neighborhood determinants of health differ in Black and non-Latino White head and neck cancer patients [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr B43.

Abstract 2250: Tumor mutational burden: Guidelines for derivation and robustness of measurement

Abstract Tumor mutational burden (TMB) is defined as the number of somatic mutations per Mb of the genome and is used to represent accumulation of somatic mutations over the life of the tumor. Recent evidence has shown that high TMB scores are associated with improved response to immune therapies across multiple indications. However, the derivation of this score is not well characterized, nor is this score universally performed across laboratories. Here we describe our methodology for calculating TMB, which considers an estimation of true germline mutations, the efficacy of different somatic callers, and the identification of regions within a capture panel that most reliably contribute to robust TMB scoring. To accomplish this, we only include positions that fall within high confidence regions of the genome as defined by NIST GIAB datasets HG001-HG005 v3.3.2. The high confidence regions encompass 88% of the exome. Somatic variant calling is performed utilizing the tumor and normal samples using only positions where there is sufficient depth of coverage in both samples. Any remaining germline variants are excluded, leaving only somatic variants in the targeted, high-confidence regions for the final TMB calculation. TMB calculations utilizing high-confidence regions reduce the contribution of false positive variants in the mutational burden estimate. We have compared TMB scores using this approach to those provided from evaluating somatic calls from TCGA, where matched tumor/normal pairs exist. In addition, we introduce a method to determine the effect on TMB scores when in silico determination of germline variants is used to derive somatic calls when matching normal samples are not available. Defining the robustness of this score across and criteria for its derivation will help better position defining analytical range and future utility across indications and NGS panels Citation Format: Natalie Mola, Matthew Schu, Suzy Stiegelmeyer, Wendell Jones, Victor J. Weigman. Tumor mutational burden: Guidelines for derivation and robustness of measurement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2250.

Abstract 1286: Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability

Abstract Failure of the DNA mismatch repair (MMR) pathway during DNA replication in cancer leads to the increased accumulation of somatic mutations. The molecular hallmark of MMR deficiency is microsatellite instability (MSI), which presents as polymorphism of tandem nucleotide repeat lengths ubiquitously distributed throughout the genome. Furthermore, the presence of MMR-deficiency or MSI across solid tumors has been recently demonstrated to be a robust biomarker for immunotherapy response with checkpoint inhibition. We have previously developed technology to detect MSI in tumor tissue through hybrid-capture based targeted next-generation sequencing analyses of a 125 gene panel, with probes capturing mononucleotide repeat markers. Using a set of 32 MSI and 27 microsatellite stable (MSS) formalin-fixed paraffin-embedded tumor tissue specimens, we obtained 100% sensitivity and 100% specificity with a limit of detection of 20% tumor purity. The clinical importance of MSI status now requires a fast, objective, highly sensitive screening method, particularly in late-stage patients where tumor material may not be readily obtained. However, to extend this approach to a liquid biopsy panel requires technological advances to overcome the inherent challenges associated with low circulating tumor DNA (ctDNA) levels that are compounded by polymerase slippage in mononucleotide repeat regions during PCR amplification. To overcome these limitations, we applied a multifactorial error correction approach together with a novel peak finding algorithm to more accurately identify the specific mononucleotide sequences in cell-free DNA (cfDNA) analyses. By eliminating a significant majority of sequencing errors and polymerase slippage artifacts, we were able to reduce background error rates by > 90%. Combined with implementation of a distribution modeling and a peak finding algorithm, we were able to accurately sequence the mononucleotide tracts to minimize false discovery rates for cfDNA analyses. To evaluate the performance of this approach in clinical samples, for a cohort of plasma samples obtained from 13 late-stage clinical cases with matched tumor tissue data available (9 MSI and 4 MSS) together with 66 healthy donor plasma samples, we obtained 89% sensitivity and 100% specificity, with a lowest reported mutant allele fraction of 0.4%. Taken together, this integrated next-generation sequencing liquid biopsy approach is valuable for the determination of MSI status to inform immunotherapy treatment. Citation Format: Andrew Georgiadis, Derrick Wood, Derek Murphy, Sonya Parpart-Li, David Riley, Naomi Sengamalay, Marian Novak, Sian Jones, Samuel A. Angiuoli, Mark Sausen. Analytical validation of an integrated next-generation sequencing pan-cancer liquid biopsy approach for detection of microsatellite instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1286.

Abstract 3377: Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing

Abstract The accumulation of somatic mutations is a defining hallmark of cancer. Human colorectal cancers (CRC) contain thousands of mutations that are detected at frequencies greater than 1%. Some of these are “driver mutations” that have been positively selected during tumor progression. In addition, subclonal mutations—those occurring only in a fraction of malignant cells—are randomly distributed and contribute to phenotypic and morphologic heterogeneity of cancer cells within a tumor. Cells containing specific subclonal mutations may be present prior to therapy and can be positively selected and account for the rapid emergence of therapeutic resistance. The extent of subclonal mutations in cancer, however, has been difficult to quantify, as the high error rate of next-generation sequencing precludes reliable detection of mutations present in fewer than 1-5% of cells. Here, we apply the highly accurate duplex sequencing methodology to quantify the extent of subclonal mutations in CRC. We analyzed somatic mutations in genes encoding replicative DNA polymerases (POLδ and POLε) as well as in genes reported to be frequently mutated in CRC. Duplex sequencing was carried out at depths varying from 500X-30,000X and an accuracy of 10-8. We find that CRCs with DNA repair deficits harbor an unexpectedly large complement of unique subclonal mutations; indicating that the mutational diversity of CRCs has been greatly underestimated. The frequency of subclonal mutations in normal colonic mucosa and CRC is greater than reported for clonal mutations. The burden of tumor-associated subclonal mutations does not correlate with age and the spectrum of single-nucleotide substitutions is different from that in nonmalignant cells, indicating that different mechanisms of mutation accumulation are operative in normal and CRC. Our data are consistent with a mutation rate of 1.1 x 10 per base pair, indicating that each malignant cell and its daughter encoded genomic DNA differs by more 3,000 nucleotides. The linearity of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We calculate that the probability of mutations at any type is so high that it is likely that every possible somatic point mutation is present by the time a tumor is clinically detectable, and this could account for the high frequency by which tumors become resistant to therapeutic agents. Citation Format: Lawrence A. Loeb, Kaitlyn J. Loubet-Senear, Brendan F. Kohrn, Michael W. Schmitt, Mary P. Bronner, Robert A. Beckman. Extensive subclonal mutations in human colorectal cancers detected by duplex sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3377.

Oak genome reveals facets of long lifespan

Oaks are an important part of our natural and cultural heritage. Not only are they ubiquitous in our most common landscapes1 but they have also supplied human societies with invaluable services, including food and shelter, since prehistoric times2. With 450 species spread throughout Asia, Europe and America3, oaks constitute a critical global renewable resource. The longevity of oaks (several hundred years) probably underlies their emblematic cultural and historical importance. Such long-lived sessile organisms must persist in the face of a wide range of abiotic and biotic threats over their lifespans. We investigated the genomic features associated with such a long lifespan by sequencing, assembling and annotating the oak genome. We then used the growing number of whole-genome sequences for plants (including tree and herbaceous species) to investigate the parallel evolution of genomic characteristics potentially underpinning tree longevity. A further consequence of the long lifespan of trees is their accumulation of somatic mutations during mitotic divisions of stem cells present in the shoot apical meristems. Empirical4 and modelling5 approaches have shown that intra-organismal genetic heterogeneity can be selected for6 and provides direct fitness benefits in the arms race with short-lived pests and pathogens through a patchwork of intra-organismal phenotypes7. However, there is no clear proof that large-statured trees consist of a genetic mosaic of clonally distinct cell lineages within and between branches. Through this case study of oak, we demonstrate the accumulation and transmission of somatic mutations and the expansion of disease-resistance gene families in trees.